Compositions and methods related to engineered Fc constructs

ABSTRACT

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

PRIORITY

This application claims the benefit of PCT Application No.PCT/US2015/028926, filed on May 1, 2015, which claims priority to U.S.Provisional Patent Application Ser. No. 62/081,923, filed Nov. 19, 2014,the entire contents of which are hereby incorporated by reference intheir entirety.

BACKGROUND

Therapeutic proteins, e.g., therapeutic antibodies and Fc-fusionproteins, have rapidly become a clinically important drug class forpatients with immunological and inflammatory diseases.

SUMMARY OF THE INVENTION

The present invention features biologically active Fc domain-containingtherapeutic constructs. Such constructs may have desirable serumhalf-life and/or binding affinity and/or avidity for Fc receptors. Theseconstructs are useful, e.g., to reduce inflammation in a subject, topromote clearance of autoantibodies in a subject, to suppress antigenpresentation in a subject, to block an immune response, e.g., block animmune complex-based activation of the immune response in a subject, andto treat immunological and inflammatory diseases (e.g., autoimmunediseases) in a subject. The Fc constructs described herein can be usedto treat patients having immunological and inflammatory diseases withoutsignificant stimulation of immune cells.

In general, the invention features Fc constructs having 2-10 Fc domains,e.g., Fc constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains. Insome embodiments, the Fc construct includes 2-10 Fc domains, 2-5 Fcdomains, 3-5 Fc domains, 2-8 Fc domains, or 2-6 Fc domains. Theconstruct may include 2-6 (e.g., 2, 3, 4, 5, or 6) associatedpolypeptides, each polypeptide including at least one Fc domain monomer,wherein each Fc domain monomer of the construct is the same or differsby no more than 20 amino acids (e.g., no more than 15, 10 amino acids),e.g., no more than 20, 15, 10, 8, 7, 6, 5, 4, 3 or 2 amino acids, fromanother monomer of the construct. The Fc constructs described herein donot include an antigen-binding domain of an immunoglobulin. In someembodiments, the Fc construct (or an Fc domain within an Fc construct)is formed entirely or in part by association of Fc domain monomers thatare present in different polypeptides. In certain embodiments, the Fcconstruct does not include an additional domain (e.g., an IgM tailpieceor an IgA tailpiece) that promotes association of two polypeptides. Inother embodiments, covalent linkages are present in the Fc constructonly between two Fc domain monomers that join to form an Fc domain. Inother embodiments, the Fc construct does not include covalent linkagesbetween Fc domains. In still other embodiments, the Fc constructprovides for sufficient structural flexibility such that all orsubstantially all of the Fc domains in the Fc construct are capable ofsimultaneously interacting with an Fc receptor on a cell surface. Insome embodiments, the Fc construct includes at least two Fc domainsjoined through a linker (e.g., a flexible amino acid spacer). In oneembodiment, the domain monomers are different in primary sequence fromwild-type or from each other in that they have dimerization selectivitymodules.

An Fc construct of the invention can be in a pharmaceutical compositionthat includes a substantially homogenous population (e.g., at least 85%,90%, 95%, 98%, or 99% homogeneous) of the Fc construct having 2-10 Fcdomains, e.g., a construct having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fcdomains, such as those described herein. Consequently, pharmaceuticalcompositions can be produced that do not have substantial aggregation orunwanted multimerization of Fc constructs.

In one aspect, the Fc construct includes three polypeptides that formtwo Fc domains. The first polypeptide has the formula A-L-B, wherein Aincludes a first Fc domain monomer; L is a linker; and B includes asecond Fc domain monomer. The second polypeptide includes a third Fcdomain monomer, and the third polypeptide includes a fourth Fc domainmonomer. In this aspect, the first Fc domain monomer and the third Fcdomain monomer combine to form a first Fc domain. Similarly, the secondFc domain monomer and the fourth Fc domain monomer combine to form asecond Fc domain. Exemplary Fc constructs of this aspect of theinvention are illustrated in FIGS. 4 and 6.

In certain embodiments, the first Fc domain monomer and the third Fcdomain monomer include complementary dimerization selectivity modulesthat promote dimerization between these Fc domain monomers. In otherembodiments, the second Fc domain monomer and the fourth Fc domainmonomer include complementary dimerization selectivity modules thatpromote dimerization between these Fc domain monomers.

In certain embodiments, one or more of A, B, the second polypeptide, andthe third polypeptide consists of an Fc domain monomer. In oneembodiment, each of A, B, the second polypeptide, and the thirdpolypeptide consist of an Fc domain monomer.

In certain embodiments, the Fc construct can further include aheterologous moiety, e.g., a peptide, e.g., a peptide that binds a serumprotein, e.g., an albumin-binding peptide. The moiety may be joined tothe N-terminus or the carboxy-terminus of B or the third polypeptide,e.g., by way of a linker.

In certain embodiments, the Fc construct further includes an IgG C_(L)antibody constant domain and an IgG C_(H)1 antibody constant domain. TheIgG C_(H)1 antibody constant domain can be attached to the N-terminus ofA or the second polypeptide, e.g., by way of a linker.

In other embodiments, the second and third polypeptides of the Fcconstruct have the same amino acid sequence.

In another aspect, the invention features an Fc construct that includesfour polypeptides that form three Fc domains. The first polypeptide hasthe formula A-L-B, wherein A includes a first Fc domain monomer; L is alinker; and B includes a second Fc domain monomer. The secondpolypeptide has the formula A′-L′-B′, wherein A′ includes a third Fcdomain monomer; L′ is a linker; and B′ includes a fourth Fc domainmonomer. The third polypeptide includes a fifth Fc domain monomer, andthe fourth polypeptide includes a sixth Fc domain monomer. In thisaspect, A and A′ combine to form a first Fc domain, B and fifth Fcdomain monomer combine to form a second Fc domain, and B′ and sixth Fcdomain monomer combine to form a third Fc domain. An exemplary Fcconstruct of this aspect of the invention is illustrated in FIG. 5.

In certain embodiments, A and A′ each include a dimerization selectivitymodule that promotes dimerization between these Fc domain monomers. Inother embodiments, B and the fifth Fc domain monomer each include adimerization selectivity module that promotes dimerization between theseFc domain monomers. In yet other embodiments, B′ and the sixth Fc domainmonomer each include a dimerization selectivity module that promotesdimerization between these Fc domain monomers.

In certain embodiments, one or more of A, B, A′, B′, the thirdpolypeptide, and the fourth polypeptide consists of an Fc domainmonomer. In one embodiment, each of A, B, A′, B′, the third polypeptide,and the fourth polypeptide consists of an Fc domain monomer.

In certain embodiments, the Fc construct further includes an IgG C_(L)antibody constant domain and an IgG C_(H)1 antibody constant domain,wherein the IgG C_(L) antibody constant domain is attached to theN-terminus of the IgG C_(H)1 antibody constant domain by way of a linkerand the IgG C_(H)1 antibody constant domain is attached to theN-terminus of A, e.g., by way of a linker. In one embodiment, the Fcconstruct further includes a second IgG C_(L) antibody constant domainand a second IgG C_(H)1 antibody constant domain, wherein the second IgGC_(L) antibody constant domain is attached to the N-terminus of thesecond IgG C_(H)1 antibody constant domain, e.g., by way of a linker andthe second IgG C_(H)1 antibody constant domain is attached to theN-terminus of A′, e.g., by way of a linker.

In certain embodiments, the Fc construct further includes a heterologousmoiety, e.g., a peptide, e.g., an albumin-binding peptide joined to theN-terminus or C-terminus of B or B′, e.g., by way of a linker.

In other embodiments, the first and second polypeptides of the Fcconstruct have the same amino acid sequence and the third and fourthpolypeptides of the Fc construct have the same amino acid sequence.

In another aspect, the invention features an Fc construct that includestwo polypeptides. The first polypeptide has the formula A-L-B, wherein Aincludes a first Fc domain monomer; L is a linker; and B includes aserum protein-binding moiety, e.g., an albumin binding peptide. Thesecond polypeptide includes a second Fc domain monomer. In this aspect,the first Fc domain monomer and the second Fc domain monomer combine toform an Fc domain.

In certain embodiments, the first Fc domain monomer and the second Fcdomain monomer include complementary dimerization selectivity modulesthat promote dimerization between the first Fc domain monomer and thesecond Fc domain monomer.

In certain embodiments, A and the second polypeptide each consists of anFc domain monomer.

In yet another aspect, the invention features an Fc construct thatincludes two polypeptides. The first polypeptide has the formulaA-L1-B-L2-C, wherein A includes an IgG C_(L) antibody constant domain;L1 and L2 are each a linker; B includes an IgG C_(H)1 antibody constantdomain; and C includes a first Fc domain monomer. The second polypeptidehas the formula A′-L1′-B′-L2′-C′, wherein A′ includes an IgG C_(L)antibody constant domain; L1′ and L2′ are each a linker; B′ includes anIgG C_(H)1 antibody constant domain; and C′ includes a second Fc domainmonomer. In this aspect, the first Fc domain monomer and the second Fcdomain monomer combine to form an Fc domain. An exemplary Fc constructof this aspect of the invention is illustrated in FIG. 7A.

In certain embodiments, the first Fc domain monomer and the second Fcdomain monomer include dimerization selectivity modules that promotedimerization between the first Fc domain monomer and the second Fcdomain monomer.

In certain embodiments, C and C′ each consist of an Fc domain monomer.

In certain embodiments, the Fc construct further includes a serumprotein binding moiety, e.g., an albumin-binding peptide joined to theN-terminus or C-terminus of C or C′ by way of a linker.

In yet another aspect, the invention features an Fc construct thatincludes four or more polypeptides. The first polypeptide has theformula A-L1-B-L2-C, wherein A includes an IgG C_(L) antibody constantdomain; L1 and L2 are each a linker; B includes an IgG C_(H)1 antibodyconstant domain; and C includes a first Fc domain monomer. The secondpolypeptide has the formula A′-L1′-B′-L2′-C′, wherein A′ includes an IgGC_(L) antibody constant domain; L1′ and L2′ are each a linker; B′includes an IgG C_(H)1 antibody constant domain; and C′ includes asecond Fc domain monomer. In this aspect, the first Fc domain monomercombines with a third Fc domain monomer to form a first Fc domain andthe second Fc domain monomer combines with a fourth Fc domain monomer toform a second Fc domain. Additionally, the IgG C_(H)1 antibody constantdomain of the first polypeptide combines with the IgG C_(L) antibodyconstant domain of the second polypeptide and the IgG C_(H)1 antibodyconstant domain of the second polypeptide combines with the IgG C_(L)antibody constant domain of the first polypeptide to form an Fcconstruct that includes two or more Fc domains. An exemplary Fcconstruct of this aspect of the invention is illustrated in FIG. 7B.

In another aspect, the invention features an Fc construct that includestwo polypeptides. The first polypeptide includes a first Fc domainmonomer and the second polypeptide includes a second Fc domain monomer.In this aspect, the first and second Fc domain monomers combine to forman Fc domain. An exemplary Fc construct of this aspect of the inventionis illustrated in FIG. 1. Further in this aspect, the first Fc domainmonomer and the second Fc domain monomer each include a dimerizationselectivity module that promotes dimerization between the first Fcdomain monomer and the second Fc domain monomer. Exemplary Fc constructsof this embodiment are illustrated in FIGS. 2 and 3.

In certain embodiments, the first and second polypeptides each consistof an Fc domain monomer.

In certain embodiments, the Fc construct further includes a serumprotein binding moiety, e.g., an albumin-binding peptide joined to theN-terminus or C-terminus of the first or second polypeptide, e.g., byway of a linker.

In another aspect, the invention features an Fc construct that includestwo polypeptides. The first polypeptide has the formula A-L-B, wherein Aincludes a first Fc domain monomer; L is a linker; and B includes asecond Fc domain monomer. The second polypeptide has the formulaA′-L′-B′, wherein A′ includes a third Fc domain monomer; L′ is a linker;and B′ includes a fourth Fc domain monomer. In this aspect, the firstand second Fc domain monomers each include an engineered cavity intotheir respective C_(H)3 antibody constant domains and the second andfourth Fc domain monomers each include an engineered protuberance intotheir respective C_(H)3 antibody constant domains, wherein theengineered cavity and the engineered protuberance are positioned to forma protuberance-into-cavity pair. Also in this aspect, the first Fcdomain monomer and the third Fc domain monomer combine to form a firstFc domain and the second Fc domain monomer and the fourth Fc domainmonomer combine to form a second Fc domain.

In certain embodiments, one or more of A, B, A′, and B′ consists of anFc domain monomer. In one embodiment, each of A, B, A′, and B′ consistsof an Fc domain monomer.

In certain embodiments, the Fc construct further includes a serumprotein binding moiety, e.g., an albumin-binding peptide joined to theN-terminus or C-terminus of B or B′, e.g., by way of a linker.

In certain embodiments, the Fc construct further includes an IgG C_(L)antibody constant domain and an IgG C_(H)1 antibody constant domain,wherein the IgG C_(L) antibody constant domain is attached to theN-terminus of the IgG C_(H)1 antibody constant domain, e.g., by way of alinker and the IgG C_(H)1 antibody constant domain is attached to theN-terminus of A by way of a linker. In one embodiment, the Fc constructfurther includes a second IgG C_(L) antibody constant domain and asecond IgG C_(H)1 antibody constant domain, wherein the second IgG C_(L)antibody constant domain is attached to the N-terminus of the second IgGC_(H)1 antibody constant domain by way of a linker and the second IgGC_(H)1 antibody constant domain is attached to the N-terminus of A′ byway of a linker.

In another aspect, the invention features an Fc construct consisting ofa) a first polypeptide having the formula A-L-B; wherein A includes orconsists of a first Fc domain monomer; L is a linker; and B includes orconsists of a second Fc domain monomer; b) a second polypeptide havingthe formula A′-L′-B′; wherein A′ includes or consists of a third Fcdomain monomer; L′ is a linker; and B′ includes or consists of a fourthFc domain monomer; c) a third polypeptide that includes or consists of afifth Fc domain monomer; and d) a fourth polypeptide that includes orconsists of a sixth Fc domain monomer. A of the first polypeptide and A′of the second polypeptide combine to form a first Fc domain; B of thefirst polypeptide and the fifth Fc domain monomer combine to form asecond Fc domain; and B′ of the second polypeptide and the sixth Fcdomain monomer combine to form a third Fc domain. Each of the first andthird Fc domain monomers includes complementary dimerization selectivitymodules that promote dimerization between the first Fc domain monomerand the third Fc domain monomer, each of the second and fifth Fc domainmonomers includes complementary dimerization selectivity modules thatpromote dimerization between the second Fc domain monomer and the fifthFc domain monomer, and each of the fourth and sixth Fc domain monomersincludes complementary dimerization selectivity modules that promotedimerization between the fourth Fc domain monomer and the sixth Fcdomain monomer; wherein the Fc construct contains no more than three Fcdomains.

In some embodiments of this aspect, either the first Fc domain monomeror the third Fc domain monomer includes a negatively-charged amino acidsubstitution, and the other Fc domain monomer includes apositively-charged amino acid substitution, either the second and fourthFc domain monomers or the fifth and sixth Fc domain monomers include anengineered protuberance, and the other Fc domain monomers include anengineered cavity. In some embodiments, linker L1, L2, L1′, and/or L2′is 3-200 amino acids in length. In some embodiments, linker L and/or L′consists of the sequence of any one of SEQ ID NOs: 1, 2, and 3.

In another aspect, the invention features an Fc construct consisting ofa) a first polypeptide having the formula A-L1-B-L2-C; wherein Aincludes or consists of a first Fc domain monomer; L1 is a linker; Bincludes or consists of a second Fc domain monomer; L2 is a linker; andC includes or consists of a third Fc domain monomer; and b) a secondpolypeptide having the formula A′-L1′-B′-L2′-C′; wherein A′ includes orconsists of a fourth Fc domain monomer; L1′ is a linker; B′ includes orconsists of a fifth Fc domain monomer; L2′ is a linker; and C′ includesor consists of a sixth Fc domain monomer; c) a third polypeptide thatincludes or consists of a seventh Fc domain monomer; d) a fourthpolypeptide that includes or consists of a eighth Fc domain monomer; e)a fifth polypeptide that includes or consists of a ninth Fc domainmonomer; and f) a sixth polypeptide that includes or consists of a tenthFc domain monomer. A of the first polypeptide and the seventh Fc domainmonomer combine to form a first Fc domain; B of the first polypeptideand B′ of the second polypeptide combine to form a second Fc domain; Cof the first polypeptide and the eighth Fc domain monomer combine toform a third Fc domain, A′ of the second polypeptide and the ninth Fcdomain monomer combine to form a fourth Fc domain, and C′ of the secondpolypeptide and the tenth Fc domain monomer combine to form a fifth Fcdomain. Each of the first and seventh Fc domain monomers includescomplementary dimerization selectivity modules that promote dimerizationbetween the first Fc domain monomer and the seventh Fc domain monomer,each of the second and fifth Fc domain monomers includes complementarydimerization selectivity modules that promote dimerization between thesecond Fc domain monomer and the fifth Fc domain monomer, each of thethird and eighth Fc domain monomers includes complementary dimerizationselectivity modules that promote dimerization between the third Fcdomain monomer and the eighth Fc domain monomer; each of the fourth andninth Fc domain monomers includes complementary dimerization selectivitymodules that promote dimerization between the fourth Fc domain monomerand the ninth Fc domain monomer; and each of the sixth and tenth Fcdomain monomers includes complementary dimerization selectivity modulesthat promote dimerization between the sixth domain monomer and the tenthFc domain monomer; wherein the Fc construct contains no more than fiveFc domains.

In some embodiments of this aspect, each of the first, third, fourth,and sixth Fc domain monomers includes an engineered protuberance, thesecond Fc domain monomer includes a negatively-charged amino acidsubstitution, the fifth Fc domain monomer includes a positively-chargedamino acid substitution, and each of the seventh, eighth, ninth, andtenth Fc domain monomers includes an engineered cavity. In someembodiments, linker L1, L2, L1′, and/or L2′ is 3-200 amino acids inlength. In some embodiments, linker L1, L2, L1′, and/or L2′ consists ofthe sequence of any one of SEQ ID NOs: 1, 2, and 3.

In another aspect, the invention features an Fc construct consisting ofa) a first polypeptide having the formula A-L1-B-L2-C; wherein Aincludes or consists of a first Fc domain monomer; L1 is a linker; Bincludes or consists of a second Fc domain monomer; L2 is a linker; andC includes or consists of a third Fc domain monomer; and b) a secondpolypeptide having the formula A′-L1′-B′-L2′-C′; wherein A′ includes orconsists of a fourth Fc domain monomer; L1′ is a linker; B′ includes orconsists of a fifth Fc domain monomer; L2′ is a linker; and C′ includesor consists of a sixth Fc domain monomer; c) a third polypeptide thatincludes or consists of a seventh Fc domain monomer; d) a fourthpolypeptide that includes or consists of a eighth Fc domain monomer; e)a fifth polypeptide that includes or consists of a ninth Fc domainmonomer; f) a sixth polypeptide that includes or consists of a tenth Fcdomain monomer. A of the first polypeptide and A′ of the secondpolypeptide combine to form a first Fc domain; B of the firstpolypeptide and the seventh Fc domain monomer combine to form a secondFc domain; C of the first polypeptide and the eighth Fc domain monomercombine to form a third Fc domain, B′ of the second polypeptide and theninth Fc domain monomer combine to form a fourth Fc domain, and C′ ofthe second polypeptide and the tenth Fc domain monomer combine to form afifth Fc domain. Each of the first and fourth Fc domain monomersincludes complementary dimerization selectivity modules that promotedimerization between the first Fc domain monomer and the fourth Fcdomain monomer, each of the second and seventh Fc domain monomersincludes complementary dimerization selectivity modules that promotedimerization between the second Fc domain monomer and the seventh Fcdomain monomer, each of the third and eighth Fc domain monomers includescomplementary dimerization selectivity modules that promote dimerizationbetween the third Fc domain monomer and the eighth Fc domain monomer;each of the fifth and ninth Fc domain monomers includes complementarydimerization selectivity modules that promote dimerization between thefifth Fc domain monomer and the ninth Fc domain monomer; and each of thesixth and tenth Fc domain monomers includes complementary dimerizationselectivity modules that promote dimerization between the sixth domainmonomer and the tenth Fc domain monomer; wherein the Fc constructcontains no more than five Fc domains.

In some embodiments of this aspect, the first Fc domain monomer includesa negatively-charged amino acid substitution, the fourth Fc domainmonomer includes a positively-charged amino acid substitution, each ofthe second, third, fifth, and sixth Fc domain monomers includes anengineered protuberance, and each of the seventh, eighth, ninth, andtenth Fc domain monomers includes an engineered cavity. In someembodiments, linker L1, L2, L1′, and/or L2′ is 3-200 amino acids inlength. In some embodiments, linker L1, L2, L1′, and/or L2′ consists ofthe sequence of any one of SEQ ID NOs: 1, 2, and 3.

In another aspect, the invention features an Fc construct that includesone or more Fc domains, wherein the Fc construct is assembled from asingle polypeptide sequence. The polypeptide has the formula A-L-B,wherein A includes a first Fc domain monomer; L is a linker (optionallya cleavable linker with, e.g., one, two or more cleavage sites); and Bincludes a second Fc domain monomer. The linker can be an amino acidspacer of sufficient length (e.g., at least 15 amino acids, preferablyat least about 20 amino acid residues in length, e.g., 15-200 aminoacids in length) and flexibility that the first Fc domain monomer andthe second Fc domain monomer of the polypeptide combine to form an Fcdomain. In certain embodiments, the first Fc domain monomer and thesecond Fc domain monomer include complementary dimerization selectivitymodules that promote dimerization between the first Fc domain monomerand the second Fc domain monomer. Such a construct can be formed fromexpression of a single polypeptide sequence in a host cell. In oneembodiment, the polypeptide has the formula A-L1-B-L2-C, wherein Aincludes a first Fc domain monomer; L1 is a linker (optionally acleavable linker with, e.g., one, two, or more cleavage sites); Bincludes a second Fc domain monomer; L2 is a linker; and C is a third Fcdomain monomer. The linker can be an amino acid spacer of sufficientlength (e.g., at least 15 amino acids, preferably at least about 20amino acid residues in length, e.g., 15-200 amino acids in length) andflexibility that the first Fc domain monomer and the second Fc domainmonomer of the polypeptide combine to form an Fc domain. In certainembodiments, the first Fc domain monomer and the second Fc domainmonomer include complementary dimerization selectivity modules thatpromote dimerization between the first Fc domain monomer and the secondFc domain monomer. An example of an Fc construct of this embodiment,including three Fc domains, is depicted in FIG. 10.

In any of the Fc constructs described herein, the Fc domain monomers ofan Fc domain of the construct can have the same primary amino acidsequence. For example, the Fc domain monomers of an Fc domain may bothbe a wild-type sequence, or both Fc domain monomers of an Fc domain mayhave the same dimerization selectivity module, e.g., both Fc domainmonomers of an Fc domain may have identical reverse charge mutations inat least two positions within the ring of charged residues at theinterface between C_(H)3 domains.

In any of the Fc constructs described herein, the Fc domain monomers ofan Fc domain of a construct can have different sequences, e.g.,sequences that differ by no more than 20 amino acids (e.g., no more than15, 10 amino acids), e.g., no more than 20, 15, 10, 8, 7, 6, 5, 4, 3 or2 amino acids, between two Fc monomers (i.e., between the Fc domainmonomer and another monomer of the Fc construct). For example, Fcmonomer sequences of a construct described herein may be differentbecause complementary dimerization selectivity modules of any of the Fcconstructs can include an engineered cavity in the C_(H)3 antibodyconstant domain of one of the domain monomers and an engineeredprotuberance in the C_(H)3 antibody constant domain of the other of theFc domain monomers, wherein the engineered cavity and the engineeredprotuberance are positioned to form a protuberance-into-cavity pair ofFc domain monomers. Exemplary engineered cavities and protuberances areshown in Table 1. In other embodiments, the complementary dimerizationselectivity modules include an engineered (substituted)negatively-charged amino acid in the C_(H)3 antibody constant domain ofone of the domain monomers and an engineered (substituted)positively-charged amino acid in the C_(H)3 antibody constant domain ofthe other of the Fc domain monomers, wherein the negatively-chargedamino acid and the positively-charged amino acid are positioned topromote formation of an Fc domain between complementary domain monomers.Exemplary complementary amino acid changes are shown in Table 2.

In some embodiments, in addition to the dimerization selectivity modules(e.g., the engineered cavities and protuberances, or the engineeredpositively and negatively-charged amino acids (see, e.g., exemplaryamino acid changes in Tables 1 and 2)), an Fc construct described hereinmay also include additional amino acid substitutions from a wild typesequence in the Fc monomer sequences to, e.g., help to stabilize the Fcconstruct or to prevent protein aggregation.

In some embodiments, an Fc construct described herein includes 2-10 Fcdomains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 domains), wherein at least twoof the Fc domains of the construct have different dimerizationselectivity modules. For example, constructs 5, 8, 9 and 10 have atleast one Fc domain including engineered cavity and protuberance and atleast one Fc domain including complementary reverse charge mutations.

In other embodiments, one or more linker in an Fc construct describedherein is a bond.

In other embodiments, one or more linker in an Fc construct describedherein is a spacer, e.g., an amino acid spacer of 2-200 amino acids.

In certain embodiments, the amino acid spacer is a glycine and/or serinerich spacer, e.g., the spacer includes two or more motifs of thesequence GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG(SEQ ID NO: 3).

In certain embodiments, when an Fc construct includes an albumin-bindingpeptide, the albumin-binding peptide has the sequence of DICLPRWGCLW(SEQ ID NO: 28).

In other embodiments, one or more of the Fc domain monomers in the Fcconstructs described herein includes an IgG hinge domain, an IgG C_(H)2antibody constant domain, and an IgG C_(H)3 antibody constant domain.

In certain embodiments, each of the Fc domain monomers in the foregoingFc constructs includes an IgG hinge domain, an IgG C_(H)2 antibodyconstant domain, and an IgG C_(H)3 antibody constant domain. In certainembodiments, the IgG is of a subtype selected from the group consistingof IgG1, IgG2a, IgG2b, IgG3, and IgG4.

In yet another aspect, the invention features a pharmaceuticalcomposition that includes a substantially homogenous (e.g., at least85%, 90%, 95%, 97%, 98%, 99% homogeneous) population of any Fc constructdescribed herein. In one embodiment, a sterile syringe or vial qualifiedfor pharmaceutical use contains a pharmaceutical composition wherein theonly or primary active ingredient is a substantially homogenous (e.g.,at least 85%, 90%, 95%, 98%, or 99% homogeneous) population of any oneof the Fc constructs described herein. The pharmaceutical compositionmay include one or more inactive ingredients, e.g., selected from salts,detergents, surfactants, bulking agents, polymers, preservatives, andother pharmaceutical excipients. In another embodiment, thesubstantially homogenous pharmaceutical composition contains less than10%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, or less than 0.5% aggregates or unwanted multimers of the Fcconstruct.

In another aspect, the invention features a method of preparing any oneof the foregoing Fc constructs. The method includes providing a hostcell including a polynucleotide or polynucleotides encoding thepolypeptides needed to assemble the Fc construct, expressingpolypeptides in the host cell under conditions that allow for theformation of the Fc construct, and recovering (e.g., purifying) the Fcconstruct.

In some embodiments, the Fc construct is formed at least in part byassociation of Fc domain monomers that are present in differentpolypeptides. In certain embodiments, the Fc construct is formed byassociation of Fc domain monomers that are present in differentpolypeptides. In these embodiments, the Fc construct does not include anadditional domain that promotes association of two polypeptides (e.g.,an IgM tailpiece or an IgA tailpiece). In other embodiments, covalentlinkages (e.g., disulfide bridges) are present only between two Fcdomain monomers that join to form an Fc domain. In other embodiments,the Fc construct does not include covalent linkages (e.g., disulfidebridges) between Fc domains. In still other embodiments, the Fcconstruct provides for sufficient structural flexibility such that allor substantially all of the Fc domains in the Fc construct are capableof simultaneously interacting with an Fc receptor on a cell surface. Incertain examples of any of these embodiments, the Fc construct includesat least two Fc domains joined through a linker (e.g., a flexible aminoacid spacer).

In one embodiment, the Fc domain monomers of an Fc domain are found indifferent polypeptide chains that associate to form the Fc domain. Forexample, the constructs depicted in FIG. 4 and FIG. 6 have two Fcdomains including three associated polypeptides. One of the threepolypeptides includes two Fc domain monomers and the other two of thepolypeptides each includes one Fc domain monomer. The construct depictedin FIG. 5 has three Fc domains including four associated polypeptides;two of the four polypeptides have two Fc domain monomers and the othertwo of the four polypeptides each has one Fc domain monomer. The Fcconstruct depicted in FIG. 7B can have n Fc domains (where n is 2-10)including 2n polypeptides, each polypeptide including an Fc domainmonomer, an IgG C_(L) antibody constant domain, and an IgG C_(H)1antibody constant domain. The constructs depicted in FIGS. 8 and 9 eachhas five Fc domains including six associated polypeptides. Two of thesix polypeptides have three Fc domain monomers and the other four of thesix polypeptides each has one Fc domain monomer. The construct depictedin FIG. 10, has three Fc domains including two associated polypeptides.Each of the two polypeptides contains three Fc domain monomers joined ina tandem series.

In another aspect, the invention features compositions and methods forpromoting selective dimerization of Fc domain monomers. The inventionincludes an Fc domain wherein the two Fc domain monomers of the Fcdomain include identical mutations in at least two positions within thering of charged residues at the interface between C_(H)3 antibodyconstant domains. The invention also includes a method of making such anFc domain, including introducing complementary dimerization selectivitymodules having identical mutations in two Fc domain monomer sequences inat least two positions within the ring of charged residues at theinterface between C_(H)3 antibody constant domains. The interfacebetween C_(H)3 antibody constant domains consists of a hydrophobic patchsurrounded by a ring of charged residues. When one C_(H)3 antibodyconstant domain comes together with another, these charged residues pairwith residues of the opposite charge. By reversing the charge of bothmembers of two or more complementary pairs of residues, mutated Fcdomain monomers remain complementary to Fc domain monomers of the samemutated sequence, but have a lower complementarity to Fc domain monomerswithout those mutations. In this embodiment, the identical dimerizationselectivity modules promotes homodimerization. Exemplary Fc domainsinclude Fc monomers containing the double mutants K409D/D399K,K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K,E357K/K370D, or D356K/K439E. In another embodiment, an Fc domainincludes Fc monomers including quadruple mutants combining any pair ofthe double mutants, e.g., K409D/D399K/E357K/K370E. In anotherembodiment, in addition to the identical dimerization selectivitymodules, the Fc domain monomers of the Fc domain include complementarydimerization selectivity modules having non-identical mutations thatpromote specific association (e.g., engineered cavity and protuberance).As a result, the two Fc domain monomers include two dimerizationselectivity modules and remain complementary to each other, but have adecreased complementarity to other Fc domain monomers. This embodimentpromotes heterodimerization between a cavity-containing Fc domain and aprotuberance-containing Fc domain monomer. In one example, the identicalmutations in charged pair residues of both Fc domain monomers arecombined with a protuberance on one Fc domain monomer and a cavity onthe other Fc domain monomer.

In another aspect, the invention features a method of reducinginflammation in a subject in need thereof. In another aspect, theinvention features a method of promoting clearance of autoantibodies ina subject in need thereof. In another aspect, the invention features amethod of suppressing antigen presentation in a subject in need thereof.In another aspect, the invention features a method of reducing theimmune response in a subject in need thereof, e.g., reducing immunecomplex-based activation of the immune response in a subject in needthereof. These methods include administering to the subject an Fcconstruct or pharmaceutical composition described herein.

In another aspect, the invention features a method of treating aninflammatory or autoimmune or immune disease in a subject byadministering to the subject an Fc construct or pharmaceuticalcomposition described herein (e.g., any one of constructs 1-10 and 5*).Exemplary diseases include: rheumatoid arthritis (RA); systemic lupuserythematosus (SLE); ANCA-associated vasculitis; antiphospholipidantibody syndrome; autoimmune hemolytic anemia; chronic inflammatorydemyelinating neuropathy; clearance of anti-allo in transplant,anti-self in GVHD, anti-replacement, IgG therapeutics, IgG paraproteins;dermatomyositis; Goodpasture's Syndrome; organ system-targeted type IIhypersensitivity syndromes mediated through antibody-dependentcell-mediated cytotoxicity, e.g., Guillain Barre syndrome, CIDP,dermatomyositis, Felty's syndrome, antibody-mediated rejection,autoimmune thyroid disease, ulcerative colitis, autoimmune liverdisease; idiopathic thrombocytopenia purpura; Myasthenia Gravis,neuromyelitis optica; pemphigus and other autoimmune blisteringdisorders; Sjogren's Syndrome; autoimmune cytopenias and other disordersmediated through antibody-dependent phagocytosis; other FcR-dependentinflammatory syndromes, e.g., synovitis, dermatomyositis, systemicvasculitis, glomerulitis and vasculitis.

In another aspect, the invention features an Fc construct orpharmaceutical composition described herein (e.g., any one of constructs1-10 and 5*) for use in reducing inflammation in a subject in needthereof. In another aspect, the invention features an Fc construct orpharmaceutical composition described herein (e.g., any one of constructs1-10 and 5*) for use in promoting clearance of autoantibodies in asubject in need thereof. In another aspect, the invention features an Fcconstruct or pharmaceutical composition described herein (e.g., any oneof constructs 1-10 and 5*) for use in suppressing antigen presentationin a subject in need thereof. In another aspect, the invention featuresan Fc construct or pharmaceutical composition described herein (e.g.,any one of constructs 1-10 and 5*) for use in reducing the immuneresponse in a subject in need thereof, e.g., reducing immunecomplex-based activation of the immune response in a subject in needthereof.

In another aspect, the invention features an Fc construct orpharmaceutical composition described herein (e.g., any one of constructs1-10 and 5*) for use in treating an inflammatory or autoimmune or immunedisease in a subject. Exemplary diseases include: rheumatoid arthritis(RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis;antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronicinflammatory demyelinating neuropathy; clearance of anti-allo intransplant, anti-self in GVHD, anti-replacement, IgG therapeutics, IgGparaproteins; dermatomyositis; Goodpasture's Syndrome; organsystem-targeted type II hypersensitivity syndromes mediated throughantibody-dependent cell-mediated cytotoxicity, e.g., Guillain Barresyndrome, CIDP, dermatomyositis, Felty's syndrome, antibody-mediatedrejection, autoimmune thyroid disease, ulcerative colitis, autoimmuneliver disease; idiopathic thrombocytopenia purpura; Myasthenia Gravis,neuromyelitis optica; pemphigus and other autoimmune blisteringdisorders; Sjogren's Syndrome; autoimmune cytopenias and other disordersmediated through antibody-dependent phagocytosis; other FcR-dependentinflammatory syndromes, e.g., synovitis, dermatomyositis, systemicvasculitis, glomerulitis and vasculitis.

In any of the Fc constructs described herein, it is understood that theorder of the Fc domain monomers is interchangeable. For example, in apolypeptide having the formula A-L-B, the carboxy terminus of A can bejoined to the amino terminus of L, which in turn is joined at itscarboxy terminus to the amino terminus of B. Alternatively, the carboxyterminus of B can be joined to the amino terminus of L, which in turn isjoined at its carboxy terminus to the amino terminus of C. Both of theseconfigurations are encompassed by the formula A-L-B.

In a related aspect, the invention features a host cell that expressesany one of the foregoing Fc constructs. The host cell includespolynucleotides encoding the polypeptides needed to assemble the Fcconstruct, wherein the polynucleotides are expressed in the host cell.

Definitions

As used herein, the term “Fc domain monomer” refers to a polypeptidechain that includes at least a hinge domain and second and thirdantibody constant domains (C_(H)2 and C_(H)3) or functional fragmentsthereof (e.g., fragments that that capable of (i) dimerizing withanother Fc domain monomer to form an Fc domain, and (ii) binding to anFc receptor. The Fc domain monomer can be any immunoglobulin antibodyisotype, including IgG, IgE, IgM, IgA, or IgD. Additionally, the Fcdomain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, orIgG4). An Fc domain monomer does not include any portion of animmunoglobulin that is capable of acting as an antigen-recognitionregion, e.g., a variable domain or a complementarity determining region(CDR). Fc domain monomers can contain as many as ten changes from awild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 aminoacid substitutions, additions, or deletions) that alter the interactionbetween an Fc domain and an Fc receptor. Examples of suitable changesare known in the art.

As used herein, the term “Fc domain” refers to a dimer of two Fc domainmonomers that is capable of binding an Fc receptor. In the wild-type Fcdomain, the two Fc domain monomers dimerize by the interaction betweenthe two C_(H)3 antibody constant domains, as well as one or moredisulfide bonds that form between the hinge domains of the twodimerizing Fc domain monomers.

In the present invention, the term “Fc construct” refers to associatedpolypeptide chains forming between 2-10 Fc domains as described herein.Fc constructs described herein can include Fc domain monomers that havethe same or different sequences. For example, an Fc construct can havetwo Fc domains, one of which includes IgG1 or IgG1-derived Fc domainmonomers, and a second which includes IgG2 or IgG2-derived Fc domainmonomers. In another example, an Fc construct can have two Fc domains,one of which comprises a “protuberance-into-cavity pair” and a secondwhich does not comprise a “protuberance-into-cavity pair.” In thepresent invention, an Fc domain does not include a variable region of anantibody, e.g., V_(H), V_(L), CDR, or HVR. An Fc domain forms theminimum structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV.

As used herein, the term “antibody constant domain” refers to apolypeptide that corresponds to a constant region domain of an antibody(e.g., a C_(L) antibody constant domain, a C_(H)1 antibody constantdomain, a C_(H)2 antibody constant domain, or a C_(H)3 antibody constantdomain).

As used herein, the term “promote” means to encourage and to favor,e.g., to favor the formation of an Fc domain from two Fc domain monomerswhich have higher binding affinity for each other than for other,distinct Fc domain monomers. As is described herein, two Fc domainmonomers that combine to form an Fc domain can have compatible aminoacid modifications (e.g., engineered protuberances and engineeredcavities) at the interface of their respective C_(H)3 antibody constantdomains. The compatible amino acid modifications promote or favor theselective interaction of such Fc domain monomers with each otherrelative to with other Fc domain monomers which lack such amino acidmodifications or with incompatible amino acid modifications. This occursbecause, due to the amino acid modifications at the interface of the twointeracting C_(H)3 antibody constant domains, the Fc domain monomers tohave a higher affinity toward each other than to other Fc domainmonomers lacking amino acid modifications.

As used herein, the term “a dimerization selectivity module” refers to asequence of the Fc domain monomer that facilitates the favored pairingbetween two Fc domain monomers. “Complementary” dimerization selectivitymodules are dimerization selectivity modules that promote or favor theselective interaction of two Fc domain monomers with each other.Complementary dimerization selectivity modules can have the same ordifferent sequences. Exemplary complementary dimerization selectivitymodules are described herein.

As used herein, the term “engineered cavity” refers to the substitutionof at least one of the original amino acid residues in the C_(H)3antibody constant domain with a different amino acid residue having asmaller side chain volume than the original amino acid residue, thuscreating a three dimensional cavity in the C_(H)3 antibody constantdomain. The term “original amino acid residue” refers to a naturallyoccurring amino acid residue encoded by the genetic code of a wild-typeC_(H)3 antibody constant domain.

As used herein, the term “engineered protuberance” refers to thesubstitution of at least one of the original amino acid residues in theC_(H)3 antibody constant domain with a different amino acid residuehaving a larger side chain volume than the original amino acid residue,thus creating a three dimensional protuberance in the C_(H)3 antibodyconstant domain. The term “original amino acid residues” refers tonaturally occurring amino acid residues encoded by the genetic code of awild-type C_(H)3 antibody constant domain.

As used herein, the term “protuberance-into-cavity pair” describes an Fcdomain including two Fc domain monomers, wherein the first Fc domainmonomer includes an engineered cavity in its C_(H)3 antibody constantdomain, while the second Fc domain monomer includes an engineeredprotuberance in its C_(H)3 antibody constant domain. In aprotuberance-into-cavity pair, the engineered protuberance in the C_(H)3antibody constant domain of the first Fc domain monomer is positionedsuch that it interacts with the engineered cavity of the C_(H)3 antibodyconstant domain of the second Fc domain monomer without significantlyperturbing the normal association of the dimer at the inter-C_(H)3antibody constant domain interface.

As used herein, the term “joined” is used to describe the combination orattachment of two or more elements, components, or protein domains,e.g., polypeptides, by means including chemical conjugation, recombinantmeans, and chemical bonds, e.g., disulfide bonds and amide bonds. Forexample, two single polypeptides can be joined to form one contiguousprotein structure through chemical conjugation, a chemical bond, apeptide linker, or any other means of covalent linkage. In someembodiments, a first Fc domain monomer is joined to a second Fc domainmonomer by way of a peptide linker, wherein the N-terminus of thepeptide linker is joined to the C-terminus of the first Fc domainmonomer through a chemical bond, e.g., a peptide bond, and theC-terminus of the peptide linker is joined to the N-terminus of thesecond Fc domain monomer through a chemical bond, e.g., a peptide bond.In other embodiments, the N-terminus of an albumin-binding peptide isjoined to the C-terminus of the C_(H)3 antibody constant domain of an Fcdomain monomer by way of a linker in the same fashion as mentionedabove.

As used herein, the term “associated” is used to describe theinteraction, e.g., hydrogen bonding, hydrophobic interaction, or ionicinteraction, between polypeptides (or sequences within one singlepolypeptide) such that the polypeptides (or sequences within one singlepolypeptide) are positioned to form an Fc construct that has at leastone Fc domain. For example, two polypeptides, each including one Fcdomain monomer, can associate to form an Fc construct (e.g., as depictedin FIGS. 1-3). In some embodiments, three polypeptides, e.g., onepolypeptide including two Fc domain monomers and two polypeptides eachincluding one Fc domain monomer, associate to form an Fc construct thathas two Fc domains (e.g., as is shown in FIGS. 4 and 6). In someembodiments, four polypeptides, e.g., two polypeptides each includingtwo Fc domain monomers and two polypeptides each including one Fc domainmonomer, associate to form an Fc construct that has three Fc domains(e.g., as depicted in FIG. 5). In other embodiments, 2n polypeptides,e.g., each polypeptide including an Fc domain monomer, an IgG C_(L)antibody constant domain, and an IgG C_(H)1 antibody constant domainassociate to form an Fc construct that has n Fc domains (as is depictedin FIG. 7B). The two polypeptides can associate through their respectiveFc domain monomers, or through other components of the polypeptide. Forexample, in FIG. 7B, polypeptide 708 associates with polypeptide 706through its Fc domain monomer and associates with polypeptide 710through association of its C_(L) domain associating with the C_(H)1domain of polypeptide 710. The association between polypeptides does notinclude covalent interactions. For example, in FIG. 10, Fc monomersequences 1014 and 1012 within a single polypeptide associate to form anFc domain, as do Fc monomer sequences 1004 and 1006.

As used herein, the term “linker” refers to a linkage between twoelements, e.g., protein domains. A linker can be a covalent bond or aspacer. The term “bond” refers to a chemical bond, e.g., an amide bondor a disulfide bond, or any kind of bond created from a chemicalreaction, e.g., chemical conjugation. The term “spacer” refers to amoiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acidsequence (e.g., a 3-200 amino acid, 3-150 amino acid, or 3-100 aminoacid sequence) occurring between two polypeptides or polypeptide domainsto provide space and/or flexibility between the two polypeptides orpolypeptide domains. An amino acid spacer is part of the primarysequence of a polypeptide (e.g., joined to the spaced polypeptides orpolypeptide domains via the polypeptide backbone). The formation ofdisulfide bonds, e.g., between two hinge regions or two Fc domainmonomers that form an Fc domain, is not considered a linker.

As used herein, the term “cleavable linker” refers to a linkercontaining one or more elements that can be selectively cleaved, e.g.,after a construct is formed, e.g., a cleavable linker includes apolypeptide sequence that can be selectively cleaved by a protease.

As used herein, the term “albumin-binding peptide” refers to an aminoacid sequence of 12 to 16 amino acids that has affinity for andfunctions to bind serum albumin. An albumin-binding peptide can be ofdifferent origins, e.g., human, mouse, or rat. In some embodiments ofthe present invention, an albumin-binding peptide is fused to theC-terminus of an Fc domain monomer to increase the serum half-life ofthe Fc construct. An albumin-binding peptide can be fused, eitherdirectly or through a linker, to the N- or C-terminus of an Fc domainmonomer.

As used herein, the term “multimer” refers to a molecule including atleast two associated Fc constructs described herein.

As used herein, the term “polynucleotide” refers to an oligonucleotide,or nucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand. A single polynucleotide istranslated into a single polypeptide.

As used herein, the term “polypeptide” describes a single polymer inwhich the monomers are amino acid residues which are joined togetherthrough amide bonds. A polypeptide is intended to encompass any aminoacid sequence, either naturally occurring, recombinant, or syntheticallyproduced.

As used herein, the term “amino acid positions” refers to the positionnumbers of amino acids in a protein or protein domain. The amino acidpositions for antibody or Fc constructs are numbered using the Kabatnumbering system (Kabat et al., Sequences of Proteins of ImmunologicalInterest, National Institutes of Health, Bethesda, Md., ed 5, 1991).

As used herein, the term “host cell” refers to a vehicle that includesthe necessary cellular components, e.g., organelles, needed to expressproteins from their corresponding nucleic acids. The nucleic acids aretypically included in nucleic acid vectors that can be introduced intothe host cell by conventional techniques known in the art(transformation, transfection, electroporation, calcium phosphateprecipitation, direct microinjection, etc.). A host cell may be aprokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., amammalian cell (e.g., a CHO cell). As described herein, a host cell isused to express one or more polypeptides encoding desired domains whichcan then combine to form a desired Fc construct.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that contains an activeingredient as well as one or more excipients and diluents to enable theactive ingredient suitable for the method of administration. Thepharmaceutical composition of the present invention includespharmaceutically acceptable components that are compatible with the Fcconstruct. The pharmaceutical composition is typically in aqueous formfor intravenous or subcutaneous administration.

As used herein, a “substantially homogenous population” of polypeptidesor of an Fc construct is one in which at least 85% of the polypeptidesor Fc constructs in a composition (e.g., a pharmaceutical composition)have the same number of Fc domains and the same Fc domain structure. Invarious embodiments, at least 90%, 92%, 95%, 97%, 98%, 99%, or 99.5% ofthe polypeptides or Fc constructs in the composition are the same.Accordingly, a pharmaceutical composition comprising a substantiallyhomogenous population of an Fc construct is one in which at least 85% ofthe Fc constructs in the composition have the same number of Fc domainsand the same structure. A substantially homogenous population of an Fcconstruct does not include more than 10% (e.g., not more than 8%, 5%,2%, or 1%) multimers or aggregates of the Fc construct.

As used herein, the term “pharmaceutically acceptable carrier” refers toan excipient or diluent in a pharmaceutical composition. Thepharmaceutically acceptable carrier must be compatible with the otheringredients of the formulation and not deleterious to the recipient. Inthe present invention, the pharmaceutically acceptable carrier mustprovide adequate pharmaceutical stability to the Fc construct. Thenature of the carrier differs with the mode of administration. Forexample, for oral administration, a solid carrier is preferred; forintravenous administration, an aqueous solution carrier (e.g., WFI,and/or a buffered solution) is generally used.

As used herein, “therapeutically effective amount” refers to an amount,e.g., pharmaceutical dose, effective in inducing a desired biologicaleffect in a subject or patient or in treating a patient having acondition or disorder described herein. It is also to be understoodherein that a “therapeutically effective amount” may be interpreted asan amount giving a desired therapeutic effect, either taken in one doseor in any dosage or route, taken alone or in combination with othertherapeutic agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an Fc construct (construct 1) containing adimer of two wild-type (wt) Fc domain monomers (102 and 104).

FIG. 2 is an illustration of an Fc construct (construct 2) containing adimer of two Fc domain monomers. The first Fc domain monomer (202)contains a protuberance in its C_(H)3 antibody constant domain, whilethe second Fc domain monomer (204) contains a cavity in the juxtaposedposition in its C_(H)3 antibody constant domain.

FIG. 3 is an illustration of another Fc construct (construct 3). This Fcconstruct contains a dimer of two Fc domain monomers (302 and 304),wherein both Fc domain monomers contain different charged amino acids attheir C_(H)3-C_(H)3 interface than the wt sequence to promote favorableelectrostatic interaction between the two Fc domain monomers.

FIG. 4 is an illustration of an Fc construct (construct 4) containingtwo Fc domains. This construct is formed from three polypeptides. Thefirst polypeptide (402) contains two wt Fc domain monomers (404 and 406)joined in a tandem series. Each of the second and third polypeptides(408 and 410, respectively) contains a wt Fc domain monomer.

FIG. 5 is an illustration of an Fc construct (construct 5 or construct5*) containing three Fc domains formed from four polypeptides. The firstpolypeptide (502) contains one Fc domain monomer containing differentcharged amino acids at the C_(H)3-C_(H)3 interface than the wt sequence(506) joined in a tandem series with a protuberance-containing Fc domainmonomer (504). The second polypeptide (508) contains an Fc domainmonomer containing different charged amino acids at the C_(H)3-C_(H)3interface than the wt sequence (512) joined in a tandem series withanother protuberance-containing Fc domain monomer (510). The third andfourth polypeptides (514 and 516, respectively) each contain acavity-containing Fc domain monomer.

FIG. 6 is an illustration of an Fc construct (construct 6) containingtwo Fc domains formed from three polypeptides. The first polypeptide(602) contains two protuberance-containing Fc domain monomers (604 and606) joined in a tandem series, while the second and third polypeptides(608 and 610, respectively) each contain an Fc domain monomer engineeredto contain a corresponding cavity.

FIG. 7A is an illustration of another Fc construct (construct 7). ThisFc construct contains a dimer of two C_(L)-C_(H)1-Fc domain monomers(702 and 704). In this embodiment, the C_(L) antibody constant domainshave joined to the adjacent C_(H)1 antibody constant domains.

FIG. 7B is an illustration of an Fc construct (construct 8) containingmultimers of C_(L)-C_(H)1-Fc domain monomers (e.g., 706, 708, and 710)containing multiple Fc domains. In this Fc construct, the constituentpolypeptide can be the same as the constituent polypeptide in construct7. The C_(L) antibody constant domain of one Fc construct (e.g., 712)interacts with the C_(H)1 antibody constant domain of a second,neighboring Fc construct (e.g., 714).

FIG. 8 is an illustration of an Fc construct (construct 9) containingfive Fc domains formed from six polypeptides. The first and secondpolypeptides (802 and 810) each contain three Fc domain monomers (804,806, 808, and 812, 814, 816, respectively) joined in a tandem series.Specifically, in polypeptide 802 or 810, a first protuberance-containingFc domain monomer (804 or 812) is connected to a second Fc domainmonomer containing different charged amino acids at the C_(H)3-C_(H)3interface than the wt sequence (806 or 814), which is connected to athird protuberance-containing Fc domain monomer (808 or 816). The thirdthrough sixth polypeptides (818, 820, 822, and 824) each contain acavity-containing Fc domain monomer and form an Fc domain with each ofFc domain monomers 804, 808, 812 and 816, respectively.

FIG. 9 is an illustration of an Fc construct (construct 10) containingfive Fc domains formed from six polypeptides. The first and secondpolypeptides (902 and 910) each contain three Fc domain monomers (904,906, 908, and 912, 914, 916, respectively) joined in a tandem series.Specifically, in polypeptide 902 or 910, a first protuberance-containingFc domain monomer (904 or 912) is connected to a secondprotuberance-containing Fc domain monomer (906 or 914), which isconnected to a third Fc domain monomer containing different chargedamino acids at the C_(H)3-C_(H)3 interface than the wt sequence (908 or916). The third through sixth polypeptides (918, 920, 922, and 924) eachcontain a cavity-containing Fc domain monomer and form an Fc domain witheach of Fc domain monomers 904, 906, 912 and 914, respectively.

FIG. 10 is an illustration of an Fc construct (construct 11) containingthree Fc domains formed from two polypeptides of identical sequence. Thetwo polypeptides (1002 and 1010) each contain three Fc domain monomers(1004, 1006, 1008, and 1012, 1014, 1016, respectively) joined in atandem series. Specifically, each polypeptide contains a firstprotuberance-containing Fc domain monomer (1004 or 1012) connected to asecond cavity-containing Fc domain monomer (1006 or 1014), which isconnected to a third Fc domain monomer with different charged aminoacids at the C_(H)3-C_(H)3 interface than the wt sequence (1008, or1016). Fc domain monomers 1008 and 1016 associate to form a first Fcdomain; Fc domain monomers 1004 and 1006 associate to form a second Fcdomain; and Fc domain monomers 1012 and 1014 associate to form a thirdFc domain. Construct 11 can be formed from expression of a singlepolypeptide sequence in a host cell.

FIGS. 11A-11B show reducing and non-reducing SDS-PAGE of construct 4,respectively.

FIGS. 12A-12B show reducing and non-reducing SDS-PAGE of construct 6,respectively.

FIG. 13 is an SDS-PAGE of construct 5 and a table showing thepercentages of the expressed protein having three Fc domains (trimer),two Fc domains (dimer), or one Fc domain (monomer) before and afterconstruct 5 purification.

FIGS. 14A and 14B show THP-1 monocyte activation (FIG. 14A) and blocking(FIG. 14B) assays using constructs 1, 5, and 6.

FIG. 15 shows effects of IVIG and constructs 5 and 6 in a K/B×N model ofrheumatoid arthritis.

FIG. 16 shows effects of IVIG and constructs 5 and 6 in a chronic ITPmodel.

FIG. 17 shows inhibition of phagocytosis by IVIg or Construct 5 in THP-1monocytic cells.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic proteins that include Fc domains of IgG can be used to treatinflammation and immunological and inflammatory diseases. The presentinvention features compositions and methods for preparing various Fcconstructs containing two or more (e.g., 2-10) Fc domains.

I. Fc Domain Monomers

An Fc domain monomer includes a hinge domain, a C_(H)2 antibody constantdomain, and a C_(H)3 antibody constant domain. The Fc domain monomer canbe of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fcdomain monomer may also be of any immunoglobulin antibody isotype (e.g.,IgG1, IgG2a, IgG2b, IgG3, or IgG4). A dimer of Fc domain monomers is anFc domain (further defined herein) that can bind to an Fc receptor,e.g., FcγRIIIa, which is a receptor located on the surface ofleukocytes. In the present invention, the C_(H)3 antibody constantdomain of an Fc domain monomer may contain amino acid substitutions atthe interface of the C_(H)3-C_(H)3 antibody constant domains to promotetheir association with each other. In some embodiments, an Fc domainmonomer includes two other constant domains, e.g., C_(L) and C_(H)1antibody constant domains, attached to the N-terminus (FIG. 7). In otherembodiments, an Fc domain monomer includes an additional moiety, e.g.,an albumin-binding peptide, attached to the C-terminus. In the presentinvention, an Fc domain monomer does not contain any type of antibodyvariable region, e.g., V_(H), V_(L), a complementarity determiningregion (CDR), or a hypervariable region (HVR).

II. Fc Domains

As defined herein, an Fc domain includes two Fc domain monomers that aredimerized by the interaction between the C_(H)3 antibody constantdomains. In the present invention, an Fc domain does not include avariable region of an antibody, e.g., V_(H), V_(L), CDR, or HVR. An Fcdomain forms the minimum structure that binds to an Fc receptor, e.g.,FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV.

III. Dimerization Selectivity Modules

In the present invention, a dimerization selectivity module is the partof the Fc domain monomer that facilitates the preferred pairing of twoFc domain monomers to form an Fc domain. Specifically, a dimerizationselectivity module is that part of the C_(H)3 antibody constant domainof an Fc domain monomer which includes amino acid substitutionspositioned at the interface between interacting C_(H)3 antibody constantdomains of two Fc monomers. In a dimerization selectivity module, theamino acid substitutions make favorable the dimerization of the twoC_(H)3 antibody constant domains as a result of the compatibility ofamino acids chosen for those substitutions. The ultimate formation ofthe favored Fc domain is selective over other Fc domains which form fromFc domain monomers lacking dimerization selectivity modules or withincompatible amino acid substitutions in the dimerization selectivitymodules. This type of amino acid substitution can be made usingconventional molecular cloning techniques well-known in the art, such asQuikChange® mutagenesis.

In some embodiments, a dimerization selectivity module includes anengineered cavity (described further herein) in the C_(H)3 antibodyconstant domain. In other embodiments, a dimerization selectivity moduleincludes an engineered protuberance (described further herein) in theC_(H)3 antibody constant domain. To selectively form an Fc domain, twoFc domain monomers with compatible dimerization selectivity modules,e.g., one C_(H)3 antibody constant domain containing an engineeredcavity and the other C_(H)3 antibody constant domain containing anengineered protuberance, combine to form a protuberance-into-cavity pairof Fc domain monomers.

In other embodiments, an Fc domain monomer with a dimerizationselectivity module containing positively-charged amino acidsubstitutions and an Fc domain monomer with a dimerization selectivitymodule containing negatively-charged amino acid substitutions mayselectively combine to form an Fc domain through the favorableelectrostatic steering (described further herein) of the charged aminoacids. Specific dimerization selectivity modules are further listed,without limitation, in Tables 1 and 2 described further below.

In other embodiments, two Fc domain monomers include dimerizationselectivity modules containing identical reverse charge mutations in atleast two positions within the ring of charged residues at the interfacebetween C_(H)3 domains. By reversing the charge of both members of twoor more complementary pairs of residues in the two Fc domain monomers,mutated Fc domain monomers remain complementary to Fc domain monomers ofthe same mutated sequence, but have a lower complementarity to Fc domainmonomers without those mutations. In one embodiment, an Fc domainincludes Fc monomers including the double mutants K409D/D399K,K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K,E357K/K370D, or D356K/K439E. In another embodiment, an Fc domainincludes Fc monomers including quadruple mutants combining any pair ofthe double mutants, e.g., K409D/D399K/E357K/K370E.

The formation of such Fc domains is promoted by the compatible aminoacid substitutions in the C_(H)3 antibody constant domains. Twodimerization selectivity modules containing incompatible amino acidsubstitutions, e.g., both containing engineered cavities, bothcontaining engineered protuberances, or both containing the same chargedamino acids at the C_(H)3-C_(H)3 interface, will not promote theformation of an Fc domain.

Furthermore, other methods used to promote the formation of Fc domainswith defined Fc domain monomers include, without limitation, the LUZ-Yapproach (U.S. Patent Application Publication No. WO2011034605) whichincludes C-terminal fusion of a monomer α-helices of a leucine zipper toeach of the Fc domain monomers to allow heterodimer formation, as wellas strand-exchange engineered domain (SEED) body approach (Davis et al.,Protein Eng Des Sel. 23:195-202, 2010) that generates Fc domain withheterodimeric Fc domain monomers each including alternating segments ofIgA and IgG C_(H)3 sequences.

IV. Engineered Cavities and Engineered Protuberances

The use of engineered cavities and engineered protuberances (or the“knob-into-hole” strategy) is described by Carter and co-workers(Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J MolBiol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681,1998). The knob and hole interaction favors heterodimer formation,whereas the knob-knob and the hole-hole interaction hinder homodimerformation due to steric clash and deletion of favorable interactions.The “knob-into-hole” technique is also disclosed in U.S. Pat. No.5,731,168.

In the present invention, engineered cavities and engineeredprotuberances are used in the preparation of the Fc constructs describedherein. An engineered cavity is a void that is created when an originalamino acid in a protein is replaced with a different amino acid having asmaller side-chain volume. An engineered protuberance is a bump that iscreated when an original amino acid in a protein is replaced with adifferent amino acid having a larger side-chain volume. Specifically,the amino acid being replaced is in the C_(H)3 antibody constant domainof an Fc domain monomer and is involved in the dimerization of two Fcdomain monomers. In some embodiments, an engineered cavity in one C_(H)3antibody constant domain is created to accommodate an engineeredprotuberance in another C_(H)3 antibody constant domain, such that bothC_(H)3 antibody constant domains act as dimerization selectivity modules(described above) that promote or favor the dimerization of the two Fcdomain monomers. In other embodiments, an engineered cavity in oneC_(H)3 antibody constant domain is created to better accommodate anoriginal amino acid in another C_(H)3 antibody constant domain. In yetother embodiments, an engineered protuberance in one C_(H)3 antibodyconstant domain is created to form additional interactions with originalamino acids in another C_(H)3 antibody constant domain.

An engineered cavity can be constructed by replacing amino acidscontaining larger side chains such as tyrosine or tryptophan with aminoacids containing smaller side chains such as alanine, valine, orthreonine. Specifically, some dimerization selectivity modules(described further above) contain engineered cavities such as Y407Vmutation in the C_(H)3 antibody constant domain. Similarly, anengineered protuberance can be constructed by replacing amino acidscontaining smaller side chains with amino acids containing larger sidechains. Specifically, some dimerization selectivity modules (describedfurther above) contain engineered protuberances such as T366W mutationin the C_(H)3 antibody constant domain. In the present invention,engineered cavities and engineered protuberances are also combined withinter-C_(H)3 domain disulfide bond engineering to enhance heterodimerformation. Specifically, the cavity Fc contains an Y349C mutation, andthe protuberance Fc contains an S354C mutation. Other engineeredcavities and engineered protuberances, in combination with eitherdisulfide bond engineering or structural calculations (mixed HA-TF) areincluded, without limitation, in Table 1.

TABLE 1 CH₃ antibody CH₃ antibody constant domain of constant domain ofStrategy Fc domain monomer 1 Fc domain monomer 2 Reference Engineeredcavities and protuberances Y407T T366Y U.S. Pat. No. 8,216,805(“knob-into-hole”) Y407A T366W U.S. Pat. No. 8,216,805 F405A T394W U.S.Pat. No. 8,216,805 Y407T T366Y U.S. Pat. No. 8,216,805 T394S F405W U.S.Pat. No. 8,216,805 T394W:Y407T T366Y:F405A U.S. Pat. No. 8,216,805T394S:Y407A T366W:F406W U.S. Pat. No. 8,216,805 T366W:T394S F405W:Y407AU.S. Pat. No. 8,216,805 Engineered cavities and protuberancesT366S:L368A:Y407V:Y349C T366W:S354C Zeidler et al., J Immunol,(“knob-into-hole”). S-S engineering 163:1246-52, 1999 Mixed HA-TFS364H:F405A Y349T:Y394F WO2006106905

Replacing an original amino acid residue in the C_(H)3 antibody constantdomain with a different amino acid residue can be achieved by alteringthe nucleic acid encoding the original amino acid residue. The upperlimit for the number of original amino acid residues that can bereplaced is the total number of residues in the interface of the C_(H)3antibody constant domains, given that sufficient interaction at theinterface is still maintained.

V. Electrostatic Steering

Electrostatic steering is the utilization of favorable electrostaticinteractions between oppositely charged amino acids in peptides, proteindomains, and proteins to control the formation of higher ordered proteinmolecules. A method of using electrostatic steering effects to alter theinteraction of antibody domains to reduce for formation of homodimer infavor of heterodimer formation in the generation of bi-specificantibodies is disclosed in U.S. Patent Application Publication No.2014-0024111.

In the present invention, electrostatic steering is used to control thedimerization of Fc domain monomers and the formation of Fc constructs.In particular, to control the dimerization of Fc domain monomers usingelectrostatic steering, one or more amino acid residues that make up theC_(H)3-C_(H)3 interface are replaced with positively- ornegatively-charged amino acid residues such that the interaction becomeselectrostatically favorable or unfavorable depending on the specificcharged amino acids introduced. In some embodiments, apositively-charged amino acid in the interface, such as lysine,arginine, or histidine, is replaced with a negatively-charged amino acidsuch as aspartic acid or glutamic acid. In other embodiments, anegatively-charged amino acid in the interface is replaced with apositively-charged amino acid. The charged amino acids may be introducedto one of the interacting C_(H)3 antibody constant domains, or both. Byintroducing charged amino acids to the interacting C_(H)3 antibodyconstant domains, dimerization selectivity modules (described furtherabove) are created that can selectively form dimers of Fc domainmonomers as controlled by the electrostatic steering effects resultingfrom the interaction between charged amino acids.

In one particular example, to create a dimerization selectivity moduleincluding reversed charges, amino acid Asp399 in the C_(H)3 antibodyconstant domain is replaced with Lys, and amino acid Lys409 is replacedwith Asp. Heterodimerization of Fc domain monomers can be promoted byintroducing different, but compatible, mutations in the two Fc domainmonomers, such as the charge residue pairs included, without limitation,in Table 2, Homodimerization of Fc domain monomers can be promoted byintroducing the same mutations in both Fc domain monomers in a symmetricfashion, such as the double mutants K409D/D399K or K392D/D399K.

TABLE 2 CH₃ antibody constant CH₃ antibody constant domain of Fc domaindomain of Fc domain monomer 1 monomer 2 Reference K409D D399K U.S. Pat.No. 2014/0024111 K409D D399R U.S. Pat. No. 2014/0024111 K409E D399K U.S.Pat. No. 2014/0024111 K409E D399R U.S. Pat. No. 2014/0024111 K392D D399KU.S. Pat. No. 2014/0024111 K392D D399R U.S. Pat. No. 2014/0024111 K392ED399K U.S. Pat. No. 2014/0024111 K392E D399R U.S. Pat. No. 2014/0024111K409D:K392D D399K:E356K Gunasekaran et al., J Biol Chem. 285:19637- 46,2010 K370E:K409D:K439E E356K:E357K:D399K Martens et al., Clin CancerRes. 12:6144-52, 2006VI. Linkers

In the present invention, a linker is used to describe a linkage orconnection between polypeptides or protein domains and/or associatednon-protein moieties. In some embodiments, a linker is a linkage orconnection between at least two Fc domain monomers, for which the linkerconnects the C-terminus of the C_(H)3 antibody constant domain of afirst Fc domain monomer to the N-terminus of the hinge domain of asecond Fc domain monomer, such that the two Fc domain monomers arejoined to each other in tandem series. In other embodiments, a linker isa linkage between an Fc domain monomer and any other protein domainsthat are attached to it. For example, a linker can attach the C-terminusof the C_(H)3 antibody constant domain of an Fc domain monomer to theN-terminus of an albumin-binding peptide. In another example, a linkercan connect the C-terminus of a C_(H)1 antibody constant domain to theN-terminus of the hinge domain of an Fc domain monomer. In yet otherembodiments, a linker can connect two individual protein domains (notincluding an Fc domain), for example, the C-terminus of a C_(L) antibodyconstant domain can be attached to the N-terminus of a C_(H)1 antibodyconstant domain by way of a linker.

A linker can be a simple covalent bond, e.g., a peptide bond, asynthetic polymer, e.g., a polyethylene glycol (PEG) polymer, or anykind of bond created from a chemical reaction, e.g. chemicalconjugation. In the case that a linker is a peptide bond, the carboxylicacid group at the C-terminus of one protein domain can react with theamino group at the N-terminus of another protein domain in acondensation reaction to form a peptide bond. Specifically, the peptidebond can be formed from synthetic means through a conventional organicchemistry reaction well-known in the art, or by natural production froma host cell, wherein a polynucleotide sequence encoding the DNAsequences of both proteins, e.g., two Fc domain monomer, in tandemseries can be directly transcribed and translated into a contiguouspolypeptide encoding both proteins by the necessary molecularmachineries, e.g., DNA polymerase and ribosome, in the host cell.

In the case that a linker is a synthetic polymer, e.g., a PEG polymer,the polymer can be functionalized with reactive chemical functionalgroups at each end to react with the terminal amino acids at theconnecting ends of two proteins.

In the case that a linker (except peptide bond mentioned above) is madefrom a chemical reaction, chemical functional groups, e.g., amine,carboxylic acid, ester, azide, or other functional groups commonly usedin the art, can be attached synthetically to the C-terminus of oneprotein and the N-terminus of another protein, respectively. The twofunctional groups can then react to through synthetic chemistry means toform a chemical bond, thus connecting the two proteins together. Suchchemical conjugation procedures are routine for those skilled in theart.

Spacer

In the present invention, a linker between two Fc domain monomers can bean amino acid spacer including 3-200 amino acids. Suitable peptidespacers are known in the art, and include, for example, peptide linkerscontaining flexible amino acid residues such as glycine and serine. Incertain embodiments, a spacer can contain motifs, e.g., multiple orrepeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2),or SGGG (SEQ ID NO: 3). In certain embodiments, a spacer can contain 2to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 4),GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO:7), or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments, aspacer can contain 3 to 12 amino acids including motifs of GGS, e.g.,GGS, GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), and GGSGGSGGSGGS(SEQ ID NO: 11). In yet other embodiments, a spacer can contain 4 to 12amino acids including motifs of GGSG (SEQ ID NO: 12), e.g., GGSG (SEQ IDNO: 13), GGSGGGSG (SEQ ID NO: 14), or GGSGGGSGGGSG (SEQ ID NO: 15). Inother embodiments, a spacer can contain motifs of GGGGS (SEQ ID NO: 16),e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 17). In other embodiments, a spacercan also contain amino acids other than glycine and serine, e.g.,GENLYFQSGG (SEQ ID NO: 18), SACYCELS (SEQ ID NO: 19), RSIAT (SEQ ID NO:20), RPACKIPNDLKQKVMNH (SEQ ID NO: 21),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 22), AAANSSIDLISVPVDSR(SEQ ID NO: 23), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:24). In certain embodiments in the present invention, a 12- or 20-aminoacid peptide spacer is used to connect two Fc domain monomers in tandemseries (FIGS. 4-6), the 12- and 20-amino acid peptide spacers consistingof sequences GGGSGGGSGGGS (SEQ ID NO: 25) and SGGGSGGGSGGGSGGGSGGG (SEQID NO: 26), respectively. In other embodiments, an 18-amino acid peptidespacer consisting of sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 27) is usedto connect C_(L) and C_(H)1 antibody constant domains (FIG. 7A-7B).

VII. Serum Protein-Binding Peptides

Binding to serum protein peptides can improve the pharmacokinetics ofprotein pharmaceuticals, and in particular the Fc constructs describedhere may be fused with serum protein-binding peptides

As one example, albumin-binding peptides that can be used in the methodsand compositions described here are generally known in the art. In oneembodiment, the albumin binding peptide includes the sequenceDICLPRWGCLW (SEQ ID NO: 28)

In the present invention, albumin-binding peptides may be attached tothe N- or C-terminus of certain polypeptides in the Fc construct. In oneembodiment, an albumin-binding peptide may be attached to the C-terminusof one or more polypeptides in constructs 1, 2, 3, or 7A (FIGS. 1, 2, 3,and 7A, respectively). In another embodiment, an albumin-binding peptidecan be fused to the C-terminus of the polypeptide encoding two Fc domainmonomers linked in tandem series in constructs 4, 5, and 6 (FIGS. 4, 5,and 6, respectively). In yet another embodiment, an albumin-bindingpeptide can be attached to the C-terminus of Fc domain monomer which isjoined to the second Fc domain monomer in the polypeptide encoding thetwo Fc domain monomers linked in tandem series, as shown in constructs 4and 6 (FIGS. 4 and 6, respectively). Albumin-binding peptides can befused genetically to Fc constructs or attached to Fc constructs throughchemical means, e.g., chemical conjugation. If desired, a spacer can beinserted between the Fc construct and the albumin-binding peptide.Without being bound to a theory, it is expected that inclusion of analbumin-binding peptide in an Fc construct of the invention may lead toprolonged retention of the therapeutic protein through its binding toserum albumin.

VIII. Fc Constructs

In general, the invention features Fc constructs having 2-10 Fc domains.These may have greater binding affinity and/or avidity than a singlewild-type Fc domain for an Fc receptor, e.g., FcγRIIIa. The inventiondiscloses methods of engineering amino acids at the interface of twointeracting C_(H)3 antibody constant domains such that the two Fc domainmonomers of an Fc domain selectively form a dimer with each other, thuspreventing the formation of unwanted multimers or aggregates. An Fcconstruct includes an even number of Fc domain monomers, with each pairof Fc domain monomers forming an Fc domain. An Fc construct includes, ata minimum, one functional Fc domain formed from a dimer of two Fc domainmonomers.

In some embodiments, an Fc construct contains one Fc domain including adimer of two Fc domain monomers (FIGS. 1-3 and 7A). The interactingC_(H)3 antibody constant domains may be unmodified (FIG. 1) or maycontain amino acid substitutions at their interface. Specifically, theamino acid substitutions can be engineered cavities (FIG. 2), engineeredprotuberances (FIG. 2), or charged amino acids (FIG. 3).

In other embodiments, an Fc construct contains two Fc domains (FIGS. 4and 6) formed from three polypeptides. The first polypeptide containstwo Fc domain monomers joined in tandem series joined by way of alinker, and the second and third polypeptides contain one Fc domainmonomer. The second and third polypeptides may be the same polypeptideor may be different polypeptides. FIG. 4 depicts an example of such anFc construct. The first polypeptide contains two wild-type Fc domainmonomers joined in tandem series by way of a linker, and the second andthird polypeptides each contain one wild-type Fc domain monomer. One ofthe Fc domain monomers in the first polypeptide forms a first Fc domainwith the second polypeptide, while the other Fc domain monomer in thefirst polypeptide forms a second Fc domain with the third polypeptide.The second and third polypeptides are not attached or linked to eachother. FIG. 6 depicts a similar Fc construct to that of FIG. 4. In FIG.6, the Fc domain monomers in the first polypeptide both containengineered protuberances in the C_(H)3 antibody constant domains, whilethe second and third polypeptides contain engineered cavities in theC_(H)3 antibody constant domains. The engineeredprotuberance-into-cavity C_(H)3-C_(H)3 interface favors the formation ofheterodimers of Fc domain monomers and prevents the uncontrolledformation of unwanted multimers. As described further herein, in Example4, dimerization selectivity modules including engineered C_(H)3 antibodyconstant domains prevent the formation of unwanted multimers that areseen in Example 3, which describes Fc construct formation from Fc domainmonomers lacking dimerization selectivity modules.

Furthermore, in other embodiments, an Fc construct can contain three Fcdomains formed from four polypeptides (FIG. 5). The first and secondpolypeptides can be the same or different, as can the third and fourthpolypeptides. In this example, the first and second polypeptides bothencode two Fc domain monomers connected by way of a linker in tandemseries, wherein one Fc domain monomer contains charged amino acidsubstitutions in the C_(H)3 antibody constant domain while the other Fcdomain monomer contains a protuberance in the C_(H)3 antibody constantdomain. The third and fourth polypeptides both encode an Fc domainmonomer with a cavity. The first and second polypeptides form a first Fcdomain with each other through interaction of the reverse charges intheir C_(H)3 antibody constant domains. The second and third Fc domainsare formed from protuberance-into-cavity interactions between theprotuberances in the first and second polypeptides and the cavities inthe third and fourth polypeptides. Each Fc domain monomer in this Fcconstruct contains a dimerization selectivity module which promotes theformation of specific Fc domains.

In yet other embodiments, a single polypeptide can form dimers (e.g.,construct 7A; FIG. 7A) or multimers (e.g., construct 7B; FIG. 7B), notthrough interaction between C_(H)3 antibody constant domains, butthrough interaction between C_(L) constant domains and C_(H)1 constantdomains. FIG. 7B depicts an Fc construct containing multiple Fc domainsin which the C_(L) domain of one Fc domain interacts with the C_(H)1domain of a neighboring Fc domain.

In yet other embodiments, Fc constructs can contain five Fc domainsformed from six polypeptides. Two examples are depicted in FIGS. 8 and9. While these depicted Fc constructs are comprised of six polypeptides,four of the polypeptides can be encoded by the same nucleic acid, andthe remaining two polypeptides can also be encoded by the same nucleicacid. As a result, these Fc constructs can be produced by the expressionof two nucleic acids in a suitable host cell.

In another embodiment, an Fc construct containing two or more Fc domainscan be formed from two polypeptides having the same primary sequence.Such a construct can be formed from expression of a single polypeptidesequence in a host cell. An example is depicted in FIG. 10. In thisexample, a single nucleic acid is sufficient to encode an Fc constructcontaining three Fc domains. Two Fc domain monomers that are part of thesame polypeptide are permitted to form an Fc domain by the inclusion ofa flexible linker of a sufficient length and flexibility; this linkermay be a cleavable linker. This same polypeptide also contains a thirdFc domain monomer joined by way of an optional flexible linker. Thisthird Fc domain monomer is capable of joining to another Fc domainmonomer to produce the Y-shaped Fc construct depicted in FIG. 10.Formation of Fc domains can be controlled through the use ofdimerization selectivity modules, as is also depicted in FIG. 10.

IX. Host Cells and Protein Production

In the present invention, a host cell refers to a vehicle that includesthe necessary cellular components, e.g., organelles, needed to expressthe polypeptides and constructs described herein from theircorresponding nucleic acids. The nucleic acids may be included innucleic acid vectors that can be introduced into the host cell byconventional techniques known in the art (transformation, transfection,electroporation, calcium phosphate precipitation, direct microinjection,etc.). Host cells can be of either mammalian or bacterial origin.Mammalian host cells include, but are not limited to, CHO (orCHO-derived cell strains, e.g., CHO-K1, CHO-DXB11 CHO-DG44), murine hostcells (e.g., NS0, Sp2/0), VERY, HEK (e.g., HEK293), BHK, HeLa, COS,MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O andHsS78Bst cells. Host cells can also be chosen that modulate theexpression of the protein constructs, or modify and process the proteinproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of protein products. Appropriate cell linesor host systems can be chosen to ensure the correct modification andprocessing of the protein expressed.

For expression and secretion of protein products from theircorresponding DNA plasmid constructs, host cells may be transfected ortransformed with DNA controlled by appropriate expression controlelements known in the art, including promoter, enhancer, sequences,transcription terminators, polyadenylation sites, and selectablemarkers. Methods for expression of therapeutic proteins are known in theart. See, for example, Paulina Balbas, Argelia Lorence (eds.)Recombinant Gene Expression: Reviews and Protocols (Methods in MolecularBiology), Humana Press; 2nd ed. 2004 edition (Jul. 20, 2004); VladimirVoynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods andProtocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012edition (Jun. 28, 2012).

X. Purification

An Fc construct can be purified by any method known in the art ofprotein purification, for example, by chromatography (e.g., ionexchange, affinity (e.g., Protein A affinity), and size-exclusion columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. For example,an Fc construct can be isolated and purified by appropriately selectingand combining affinity columns such as Protein A column withchromatography columns, filtration, ultra filtration, salting-out anddialysis procedures (see, e.g., Process Scale Purification ofAntibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; andSubramanian (ed.) Antibodies—Volume I—Production and Purification,Kluwer Academic/Plenum Publishers, New York (2004)). In some instances,an Fc construct can be conjugated to marker sequences, such as a peptideto facilitate purification. An example of a marker amino acid sequenceis a hexa-histidine peptide, which binds to nickel-functionalizedagarose affinity column with micromolar affinity. Other peptide tagsuseful for purification include, but are not limited to, thehemagglutinin “HA” tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767).

For the Fc constructs, Protein A column chromatography may be employedas a purification process. Protein A ligands interact with Fc constructsthrough the Fc region, making Protein A chromatography a highlyselective capture process that is able to remove most of the host cellproteins. In the present invention, Fc constructs may be purified usingProtein A column chromatography as described in Example 2.

XI. Pharmaceutical Compositions/Preparations

The invention features pharmaceutical compositions that include one ormore Fc constructs described herein. In one embodiment, a pharmaceuticalcomposition includes a substantially homogenous population of Fcconstructs that are identical or substantially identical in structure.In various examples, the pharmaceutical composition includes asubstantially homogenous population of any one of constructs 1-10 and5*.

A therapeutic protein construct, e.g., an Fc construct, of the presentinvention can be incorporated into a pharmaceutical composition.Pharmaceutical compositions including therapeutic proteins can beformulated by methods know to those skilled in the art. Thepharmaceutical composition can be administered parenterally in the formof an injectable formulation including a sterile solution or suspensionin water or another pharmaceutically acceptable liquid. For example, thepharmaceutical composition can be formulated by suitably combining theFc construct with pharmaceutically acceptable vehicles or media, such assterile water for injection (WFI), physiological saline, emulsifier,suspension agent, surfactant, stabilizer, diluent, binder, excipient,followed by mixing in a unit dose form required for generally acceptedpharmaceutical practices. The amount of active ingredient included inthe pharmaceutical preparations is such that a suitable dose within thedesignated range is provided.

The sterile composition for injection can be formulated in accordancewith conventional pharmaceutical practices using distilled water forinjection as a vehicle. For example, physiological saline or an isotonicsolution containing glucose and other supplements such as D-sorbitol,D-mannose, D-mannitol, and sodium chloride may be used as an aqueoussolution for injection, optionally in combination with a suitablesolubilizing agent, for example, alcohol such as ethanol and polyalcoholsuch as propylene glycol or polyethylene glycol, and a nonionicsurfactant such as polysorbate 80™, HCO-50, and the like commonly knownin the art. Formulation methods for therapeutic protein products areknown in the art, see e.g., Banga (ed.) Therapeutic Peptides andProteins: Formulation, Processing and Delivery Systems (2d ed.) Taylor &Francis Group, CRC Press (2006).

XII. Dosage

The pharmaceutical compositions are administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The pharmaceutical compositions are administered in a variety of dosageforms, e.g., intravenous dosage forms, subcutaneous dosage forms, oraldosage forms such as ingestible solutions, drug release capsules, andthe like. The appropriate dosage for the individual subject depends onthe therapeutic objectives, the route of administration, and thecondition of the patient. Generally, recombinant proteins are dosed at1-200 mg/kg, e.g., 1-100 mg/kg, e.g., 20-100 mg/kg. Accordingly, it willbe necessary for a healthcare provider to tailor and titer the dosageand modify the route of administration as required to obtain the optimaltherapeutic effect.

XIII. Indications

The pharmaceutical compositions and methods of the invention are usefulto reduce inflammation in a subject, to promote clearance ofautoantibodies in a subject, to suppress antigen presentation in asubject, to reduce the immune response, e.g., to block immunecomplex-based activation of the immune response in a subject, and totreat immunological and inflammatory conditions or diseases in asubject. Exemplary conditions and diseases include rheumatoid arthritis(RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis;antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronicinflammatory demyelinating neuropathy; clearance of anti-allo intransplant, anti-self in GVHD, anti-replacement, IgG therapeutics, IgGparaproteins; dermatomyositis; Goodpasture's Syndrome; organsystem-targeted type II hypersensitivity syndromes mediated throughantibody-dependent cell-mediated cytotoxicity, e.g., Guillain Barresyndrome, CIDP, dermatomyositis, Felty's syndrome, antibody-mediatedrejection, autoimmune thyroid disease, ulcerative colitis, autoimmuneliver disease; idiopathic thrombocytopenia purpura; Myasthenia Gravis,neuromyelitis optica; pemphigus and other autoimmune blisteringdisorders; Sjogren's Syndrome; autoimmune cytopenias and other disordersmediated through antibody-dependent phagocytosis; other FcR-dependentinflammatory syndromes e.g., synovitis, dermatomyositis, systemicvasculitis, glomerulitis and vasculitis.

EXAMPLES Example 1—Design and Cloning of DNA Plasmid Constructs

A total of eight DNA plasmid constructs were used to assemble eight Fcconstructs (FIGS. 1-7B). The DNA plasmid constructs were transfectedinto human embryonic kidney (HEK) 293 cells for protein production. Theeight encoded secreted polypeptides had the general structures asdescribed below:

A. wt Fc: wild-type Fc domain monomer (FIG. 1: 102 and 104; FIG. 4: 408and 410).

B. protuberance Fc: Fc domain monomer with engineered protuberance inC_(H)3 antibody constant domain (FIG. 2: 202).

C. cavity Fc: Fc domain monomer with engineered cavity in C_(H)3antibody constant domain (FIG. 2: 204; FIG. 5: 514 and 516).

C*. cavity Fc*: Fc domain monomer with engineered cavity in C_(H)3antibody constant domain (FIG. 2: 204; FIG. 5: 514 and 516). Cavity Fc*also contains additional amino acid substitutions relative to cavity Fc.

D. charges Fc: Fc domain monomer with reversed charges in C_(H)3antibody constant domain (FIG. 3: 302 and 304).

E. wt-12-wt Fc2: Two Fc domain monomers joined in series by way of a12-amino acid GGGS peptide linker (FIG. 4: 402).

F. protuberance-20-charges Fc2: Fc domain monomer with reversed chargesin C_(H)3 antibody constant domain and Fc domain monomer with engineeredprotuberance in C_(H)3 antibody constant domain joined in series by wayof a 20-amino acid SGGG peptide linker (FIG. 5: 502 and 508).F*. protuberance-20-charges Fc2*: Fc domain monomer with reversedcharges in C_(H)3 antibody constant domain and Fc domain monomer withengineered protuberance in C_(H)3 antibody constant domain joined inseries by way of a 20-amino acid SGGG peptide linker (FIG. 5: 502 and508). Protuberance-20-charges Fc2* also contains additional amino acidsubstitutions relative to protuberance Fc.G. protuberance-20-protuberance Fc2: Two Fc domain monomers both withengineered protuberance in C_(H)3 antibody constant domain joined inseries by way of a 20-amino acid GGGS peptide linker (FIG. 6: 602).H. C_(H)C_(L) Fc+: Fc domain monomer with C_(H)1 and C_(L) constantdomains attached to the hinge domain (FIG. 7A: 702 and 704; FIG. 7B:706, 708, 710, 712, 714, and 716). The C_(L) constant domain is attachedby way of an 18 amino acid GGGS peptide linker to a C_(H)1 constantdomain.

Fc DNA sequences were derived from human IgG1 Fc. Protuberance, cavityand charges mutations were substituted in the parental Fc sequence. DNAencoding a leader peptide derived from the human immunoglobulin KappaLight chain was attached to the 5′ region. All but one of thepolypeptides (C_(H)C_(L) Fc+) contained this encoded peptide on theamino terminus to direct protein translocation into the endoplasmicreticulum for assembly and secretion. It will be understood that any oneof a variety of leader peptides may be used in connection with thepresent invention. The leader peptide is usually clipped off in the ERlumen. An 11 nucleotide sequence containing a 5′ terminal EcoR1 site wasadded upstream of the ATG start codon. A 30 nucleotide sequencecontaining a 3′ terminal Xho1 site was added downstream of the 3′terminal TGA translation termination codon. The DNA sequences wereoptimized for expression in mammalian cells and cloned into the pcDNA3.4mammalian expression vector.

Mutations are denoted by the wild-type amino acid residue followed bythe position using the EU Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., ed. 5, 1991) and then the replacement residue insingle-letter code. The nucleotide and amino acid sequences of secretedpolypeptides A-H described above are provided below (except for cavityFc* and protuberance-20-charges Fc2*, for which only the amino acidsequences are provided).

wt Fc SEQ ID NO: 29: 1       10         20        30        40        50|       |          |         |         |         |GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 30:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK protuberance Fc SEQ ID NO: 31:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCTGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 32:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cavity Fc SEQ ID NO: 33:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTGTACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGAGCTGCGCCGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGGTTAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 34:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cavity Fc* SEQ ID NO: 45:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVEGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK charges Fc SEQ ID NO: 35:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCGCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGAAAAGCGACGGCTCATTCTTCCTGTACAGCGACCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 36:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK wt-12-wt Fc2 SEQ ID NO: 37:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTCAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTCTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTCTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAAGGCGGGGGATCTGGGGGAGGAAGCGGAGGCGGCAGCGATAAGACCCATACCTGCCCTCCCTGTCCCGCTCCCGAACTGCTGGGGGGACCCTCCGTGTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCAGAACCCCTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGAAGATCCCGAAGTCAAGTTTAACTGGTATGTGGATGGGGTCGAGGTCCACAATGCCAAAACAAAGCCTCGGGAAGAACAGTATAACTCCACCTACAGAGTCGTCAGCGTGCTGACAGTCCTTCATCAGGATTGGCTGAATGGGAAAGAGTACAAATGTAAAGTGTCTAACAAAGCTCTGCCCGCTCCTATCGAAAAGACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCTCAGGTGTACACCCTGCCACCCTCCAGAGATGAGCTGACAAAAAATCAGGTGTCACTGACATGTCTGGTGAAAGGGTTTTATCCCTCCGACATTGCTGTGGAATGGGAATCCAATGGGCAGCCTGAAAACAATTATAAGACAACACCTCCCGTGCTGGACTCCGATGGCTCATTTTTTCTGTACTCTAAACTGACAGTGGATAAGTCCAGATGGCAGCAGGGAAATGTGTTTTCCTGCTCTGTGATGCATGAAGCTCTGCATAATCACTATACACAGAAAAGCCTGTCCCTGTCCCCCGGCAAG SEQ ID NO: 38:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKprotuberance-20-charges Fc2 SEQ ID NO: 39:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCCCCTTGCCCAGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGTCTGGGGGAGGATCAGGGGGTGGAAGTGGCGGTGGATCTGGTGGTGGAAGCGGAGGCGGCGATAAGACACACACATGCCCCCCCTGTCCAGCTCCCGAACTGCTGGGGGGACCCTCCGTGTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCAGAACCCCTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGAAGATCCCGAAGTCAAGTTTAATTGGTATGTCGATGGGGTCGAGGTGCACAATGCCAAAACAAAACCTCGGGAAGAACAGTATAACTCCACATACAGAGTGGTGTCTGTCCTCACAGTCCTGCATCAGGATTGGCTCAATGGGAAAGAGTACAAATGTAAAGTCTCTAACAAGGCTCTCCCCGCTCCGATCGAAAAGACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCTCAGGTCTACACACTGCCTCCCAGCCGGGACGAGCTGACAAAAAATCAAGTGTCTCTGACCTGCCTCGTGAAGGGCTTTTATCCCTCCGACATTGCCGTCGAGTGGGAGTCCAATGGACAGCCGGAAAACAATTATAAGACCACGCCTCCAGTGCTGAAGTCCGACGGCAGCTTCTTTCTGTACTCCGACCTGACAGTGGATAAGTCCAGATGGCAGCAAGGGAATGTGTTCTCCTGTTCCGTGATGCATGAAGCCCTCCATAATCACTATACCCAGAAAAGCCTGTCCCTGTCCCCTGGCAAG SEQ ID NO: 40:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK protuberance-20-charges Fc2* SEQ ID NO: 46:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK protuberance-20-protuberance Fc2 SEQ ID NO: 41:1       10         20        30        40        50 |       |          |        |         |         |GACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTTGCAGAGATGAACTGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGTCTGGGGGAGGATCAGGGGGTGGAAGTGGCGGTGGATCTGGTGGTGGAAGCGGAGGCGGCGATAAGACACACACATGCCCCCCCTGTCCAGCTCCCGAACTGCTGGGGGGACCCTCCGTGTTTCTGTTTCCACCTAAGCCTAAGGATACGCTCATGATCTCCAGAACCCCTGAAGTCACATGTGTGGTGGTCGATGTGTCTCATGAAGATCCCGAAGTCAAGTTTAACTGGTATGTGGATGGGGTCGAGGTCCACAATGCCAAAACAAAGCCTCGGGAAGAACAGTATAACTCCACCTACAGAGTCGTCAGCGTGCTGACAGTCCTGCATCAAGATTGGCTCAATGGGAAAGAGTATAAGTGTAAAGTCTCGAACAAAGCCCTCCCCGCTCCTATCGAAAAGACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCTCAGGTCTACACACTGCCTCCATGTCGGGACGAGCTGACAAAAAATCAGGTGTCACTGTGGTGTCTGGTGAAGGGGTTTTACCCTTCCGACATTGCTGTGGAATGGGAATCCAATGGGCAGCCTGAAAACAATTATAAGACAACACCTCCCGTGCTGGACTCCGATGGCTCATTTTTTCTGTACTCTAAACTGACAGTGGATAAGTCCAGATGGCAGCAGGGAAATGTGTTTTCCTGCTCTGTGATGCATGAAGCTCTGCATAATCACTATACACAGAAAAGCCTGTCCCTGTCCCCTGGCAAG SEQ ID NO: 42:1       10         20        30        40        50 |       |          |        |         |         |DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGGDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CHCL Fc+1       10         20        30        40        50 |       |          |        |         |         |AGGACAGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCCAGCGACGAGCAGCTCAAGTCCGGCACAGCCAGCGTGGTCTGCCTGCTGAACAACTTCTACCCCCCCGACCCCAAGGTCCAGTGGAAGCTGGACAACGCCCTGCACAGCGCCAACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGTCTAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGACGCGAGTGCGGCGGCTCTGGCGGAGGATCCGGGGGAGGATCAGGCGGCGGAAGCGGAGGCAGCGCTACCACAAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCTAGCAGCAGCCTGGCCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGAGCTGCGACAAGACCCACACGTGTCCCCCCTGCCCAGCCCCTGAACTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGCAAGTGCACAATGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCCCTCATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGACC CTGAGCCCCGGCAAASEQ ID NO: 44: 1       10         20        30        40        50 |      |          |         |         |         |RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGGGSGGGSGGGSGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

Example 2—Expression of Fc Construct Proteins

For protein expression of the Fc constructs, two of the DNA plasmidconstructs selected from A-H described in Example 1 were transfectedinto EXP1293 cells (LifeTechnologies). Liposome transfection was used tointroduce plasmid DNA into EXP1293 cells. The total amount oftransfected plasmid constructs was fixed whereas the ratio of differentplasmid constructs was varied to maximize the yield of desiredconstructs (see Table 3 below). For each Fc construct, the ratio (bymass) of the two transfected DNA plasmid constructs is shown in Table 3.Illustrations of the constructs are shown FIGS. 1-7B.

After protein expression, the expressed constructs were purified fromthe cell culture supernatant by Protein A-based affinity columnchromatography. Media supernatants were loaded onto a Poros MabCapture A(LifeTechnologies) column using an AKTA Avant preparative chromatographysystem (GE Healthcare Life Sciences). Captured Fc constructs were thenwashed with phosphate buffered saline (low-salt wash) followed byphosphate buffered saline supplemented with 500 mM NaCl (high-saltwash). Fc constructs are eluted with 100 mM glycine, 150 mM NaCl, pH 3buffer. The protein solution emerging from the column is neutralized byaddition of 1M TRIS pH 7.4 to a final concentration of 100 mM. The Fcconstructs were further fractionated by ion exchange chromatographyusing Poros® XS resin (Applied Biosciences Cat. #4404336). The columnwas pre-equilibrated with 10 mM MES, pH 6 (buffer A), and the sample waseluted with a gradient against 10 mM MES, 500 mM sodium chloride, pH 6(buffer B).

We obtained a total of seven Fc constructs (see Table 3 below and FIGS.1-7B). Purified Fc constructs were analyzed by SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) under both reducing andnon-reducing conditions followed by Coomassie Blue staining to confirmthe presence of protein bands of expected size.

TABLE 3 Ratio of Approx. MW in Approx. MW in Plasmids kDa (reducing KDa(non-reducing Construct Plasmids Transfected A:B SDS-Page) SDS-Page) 1A: Wt Fc n/a 25 50 2 A: Protuberance Fc 1:1 25 50 B: Cavity Fc 3 A:Charges Fc 25 50 4 A: Wt-12-Wt Fc2 1:2 25, 50 100 B: Wt FC 5 A:Protuberance-20-Charges Fc2 2:1 25, 50 150 B: Cavity Fc 5* A:Protuberance-20-Charges Fc2* 2:1 25, 50 150 B: Cavity Fc* 6 A:Protuberance-20-Protuberance Fc2 1:1 25, 50 100 B: Cavity Fc 7 and 8 A:Ch, Cl, Fc+ n/a 50 100

Example 3—Preparation and SDS-Page Analysis of Construct 4

Two DNA plasmid constructs, wt-12-wt Fc2 (DNA plasmid construct E inExample 1) and wt Fc (DNA plasmid construct A in Example 1), were usedto express construct 4 (FIG. 4). The two plasmid constructs weretransfected into HEK 293 cells for protein expression and purificationas described in Example 2. FIG. 11A-B shows the reducing andnon-reducing SDS-PAGE of construct 4. On reducing SDS-PAGE (FIG. 11A),we observed a band at approximately 25 kDa (lanes 2 and 3, FIG. 11A)corresponding to the wt Fc domain monomer and a band at 50 kDacorresponding to the wt-12-wt Fc2 tandem dimer (lanes 1-3, FIG. 11A). Onnon-reducing SDS-PAGE (FIG. 11B), lanes 2 and 3 each contain the finalprotein product of construct 4 in higher (½) and lower (⅓) proteinamounts, respectively. We observed one major band at approximately 100kDa corresponding to the association of wt-12-wt Fc2 tandem dimer withtwo wt Fc domain monomers to form construct 4, and another major band ofapproximately equal signal intensity at approximately 50 kDacorresponding to free wt-12-wt Fc2 tandem dimer that is not joined withwt Fc domain monomers.

In addition, we observed higher molecular weight bands at approximately150 kDa, 200 kDa and 250 kDa (lanes 2 and 3, FIG. 11B) corresponding tomultimers of wt-12-wt Fc2 and wt Fc domain monomer.

Example 4—Preparation and SDS-Page Analysis of Construct 6

Two plasmid constructs, protuberance-20-protuberance Fc2 (DNA plasmidconstruct G in Example 1) and cavity Fc (DNA plasmid construct C inExample 1), were used to express construct 6 (FIG. 6). The two plasmidconstructs were transfected into HEK 293 cells for protein expressionand purification as described in Example 2. FIGS. 12A-12B show thereducing and non-reducing SDS-PAGE of construct 6. On reducing SDS-PAGE(FIG. 12A), we observed a band at approximately 25 kDa (lanes 2 and 3,FIG. 12A) corresponding to the cavity Fc domain monomer and a band at 50kDa corresponding to the protuberance-20-protuberance Fc2 tandem dimer(lanes 1-3, FIG. 12A). On non-reducing SDS-PAGE (FIG. 12B), lanes 2 and3 each contain the final protein product of construct 6 in higher (½)and lower (⅓) protein amounts. We observed one major band atapproximately 100 kDa corresponding to the association of theprotuberance-20-protuberance Fc2 tandem dimer with two cavity Fc domainmonomers and a minor band of weaker signal intensity at approximately 50kDa corresponding to free protuberance-20-protuberance Fc2 tandem dimerthat was not combined with any cavity Fc domain monomer.

A similar experiment was performed with construct 5 (FIG. 13). Twoplasmid constructs, protuberance-20-charges Fc2 (DNA plasmid construct Fin Example 1) and cavity Fc (DNA plasmid construct C in Example 1), wereused to express construct 5 (FIG. 5). The two plasmid constructs weretransfected into EXP1293 cells at empirically determined ratios bycationic lipid transfection. The transfected cultures are incubated incell culture media for 6-8 days. After this time, the cells were removedby centrifugation. The supernatant (media, lane 1 of FIG. 13) containsconstruct 5 which was secreted by the transfected cells into the media.There are also contaminating host cell proteins in the media. Construct5 was purified from the media by Protein-A affinity chromatography. Atthis point, the media contained the desired construct 5 having three Fcdomains (trimer) as well as a some proportion of misassembled proteinshaving two Fc domains (dimer, about 10-15%) and one Fc domain (monomer,5-10%). There was also a small amount of contaminating host cellproteins still present. The Protein A column eluate was bufferexchanged, concentrated, and fractionated by Strong Cation Exchange(SCX) chromatography. Briefly, construct 5 was bound to the SCX columnand then eluted with a salt and pH gradient. This step enabledseparation of the desired construct 5 having three Fc domains from mostof the misassembled proteins having two or one Fc domain, from construct5 having unwanted post translational modifications, and fromcontaminating host cell proteins. After another round of concentrationand buffer exchange, a pure, final protein product of construct 5 wasobtained (pure, lane 2 of FIG. 13).

FIG. 13 depicts an SDS-PAGE of media obtained from cultured host cellsengineered to express construct 5 (lane 1), and of purified construct 5(lane 2). Also shown is a table showing the percentages of the majorbands of the SDS-PAGE for each sample. In the media sample (lane 1), amajor band at approximately 150 kDa was observed, corresponding to thefinal protein product of construct 5 having three Fc domains. The mediasample also contained a minor band of weaker signal intensity at 100 kDacorresponding to a protein having two Fc domains, and a second minorband of weakest signal intensity at 50 kDa corresponding a proteinhaving one Fc domain. After purification (lane 2), the major band atapproximately 150 kDa, corresponding to the final protein product ofconstruct 5 having three Fc domains is enriched. Quantification of thesignal intensities of the protein bands on the SDS-PAGE of construct 5showed that, in the culture media, before protein purification, about79% of the total protein was the desired protein product of construct 5.After protein purification, a substantially homogenous population ofconstruct 5 having about 95% purity was obtained.

These findings demonstrate that the selectivity dimerization modulecontaining either an engineered protuberance or an engineered cavity inthe C_(H)3 antibody constant domain reduces self-association andprevents uncontrolled Fc-mediated aggregate or multimer formation,indicating that the use of dimerization selectivity modules in theconstructs described herein can be used to produce substantiallyhomogenous preparations of the Fc constructs. This observation hassignificant implications for advantages in manufacturing, yield, andpurity of the constructs, e.g., in order to control biological activityand potency.

Example 5—Binding Affinity and Avidity

The binding of constructs to multiple Fcγ receptors was assessed usingcell-based FRET competition assays (Cisbio Bioassays). Constructs 5 and6 showed at least a ten-fold decrease in IC50 (i.e. increased binding)to FcγRIIa, FcγRIIb, and FcγRIIIa relative to the wild type Fc domain(construct 1).

Example 6—Monocyte Activation and Blocking Assays

Three Fc constructs, constructs 1, 5, and 6, containing one, three, andtwo Fc domains, respectively, were tested for their ability to activateTHP-1 monocytes on their own. IL-8 release was used as an indicator ofmonocyte activation. Constructs 1, 5, and 6 were expressed and purifiedas described in Examples 1 and 2. Each of the purified Fc constructs wasadded to THP-1 monocytes. No substantial IL-8 release was observed forany of the three constructs. The data are provided in FIG. 14A.

The same three Fc constructs were then tested for their ability toinhibit Fc receptor-mediated monocyte activation. IgG1 (100 μg/mL) wasimmobilized on a 96 well plate and used to induce IL-8 release by THP-1monocytes. Serial dilutions of constructs 1, 5 and 6 or controlsubstances (intravenous immunoglobulin (IVIg), human serum albumin(HSA), and glycine buffer) were subsequently performed in the tissueculture plate. THP-1 monocytes (1.5×10⁵ cells) were immediately addedwith thorough mixing. The cultures were incubated for 18 h and thesupernatants analyzed for IL-8. Constructs 5 and 6 were found to inhibitIL-8 release more effectively than construct 1 at low doses. The dataare provided in FIG. 14B.

Example 7—K/B×n Arthritis Model

Fc constructs 1, 5, and 6 and IVIg were tested for their ability toprotect mice from joint inflammation in the K/B×N serum transfer modelusing a method described in Anthony, Proc. Natl. Acad. Sci. U.S.A.105:19571-19578 (2008). Twelve-week old K/B×N mice weregenerated/purchased from Jackson Laboratories. A total of thirty C57BLmice were separated into five groups of six mice each. Each group wasinjected intravenously (i.v.) with 200 μl construct 6 at 0.1 g/kg, 200μl construct 5 at 0.1 g/kg, 200 μl IVIg at 0.1 g/kg, 230 μl IVIg at 1g/kg, or 200 μl phosphate-buffered saline (PBS) one hour beforeinjection of 200 μl K/B×N serum (an arthritis inducing serum) (Day 0).Inflammation was scored by clinical examination of paw swelling andankle thickness. For paw swelling, each paw was scored 0-3 (0, noswelling; 3, maximal swelling). Scores of four paws were added for totalclinical score per individual mouse. For ankle thickness, calipermeasurement was used. Each mouse was scored daily from Day 0 to Day 10.The daily average clinical score for each group of six mice was plottedin FIG. 15. As shown in FIG. 15, IVIg at 1 g/kg, construct 5 at 0.1g/kg, and construct 6 at 0.1 g/kg provided similar level of inflammationprotection. Given that constructs 5 and 6 were administered at ten-foldlower dose compared to the dose of IVIg, constructs 5 and 6 appear to bemore potent than IVIg.

Example 8—Chronic ITP Model

Constructs 1 and 5, as well as IVIg, were tested for their ability totreat mice undergoing immune thrombocytopenia (ITP). ITP was induced byan anti-platelet Ab that causes platelet depletion. Forty five C57BL/6mice (18-22 g, Charles Rivers Labs, Mass.) were injected i.p. with 1.5μg/mouse of rat anti-CD41 antibody (Ab) (clone MWReg30 BioLegendcat#133910) once daily for 4 days (on days 1, 2, 3 and 4). Five micewere injected with 1.5 μg/mouse of a rat IgG1, k isotype control Ab(BioLegend cat#400414) to determine normal platelet levels. Abs wereinjected in 100 μl of PBS. All mice were dosed once intravenously with200 μl of either saline control, IVIg at 1 g/kg, construct 1 at 0.02,0.03, 0.1, and 0.3 g/kg, and construct 5 at 0.004, 0.02, and 0.1 g/kg 2h after the third anti-CD41 Ab injection on day 3. Mice were bled on day5 (24 h after the forth anti-CD41 Ab injection) to quantitate totalplatelet levels by the VetScan Instrument. All procedures were performedin compliance with the Animal Welfare Act and with the Guide for theCare and Use of Laboratory Animals.

As shown in FIG. 16, platelet levels were significantly increased aftertherapeutic treatment with construct 5 at 0.02 and 0.1 g/kg whencompared to saline control (**** p<0.0001 by One-way ANOVA with multiplecomparisons test). Platelet levels in these groups were similar to thelevels in the normal, isotype treated-group. Therapeutic treatment withIVIg at 1 g/kg and construct 1 at 0.1 and 0.3 g/kg, also significantlyincreased platelet levels when compare to saline control (* p<0.05;**p<0.01 respectively by One-way ANOVA with multiple comparisons test)but platelet levels in these groups were lower than in the 0.02 and 0.1g/kg construct 5 treated-groups. In this model, construct 5 appears tobe about 50-fold more potent than IVIg.

Example 9—Construct 5* Shows Augmented Binding and Avidity to FcγRCompared to IVIg

Following the same protocol as described in Example 8, two plasmidconstructs, encoding protuberance-20-charges Fc2* (construct F* inExample 1) and cavity Fc* (construct C* in Example 1), were used toexpress and purify construct 5*. The binding profile of this constructto various Fc receptors was compared to that of IVIG in a fluorescenceresonance energy transfer (FRET) competitive binding assay.

Construct 5* displayed an overall binding profile to the differentFcγ-receptors similar to that of IVIg (with the lowest binding affinityobserved for FcγRIIb) but with greatly enhanced binding to all lowaffinity FcγRs when compared to IVIg. Augmented binding to FcγRcorresponds to higher avidity, which refers to the cumulative effect ofthe accumulated affinities of each individual binding interaction. IC50values for construct 5* were consistently lower than those of IVIg,indicating striking increases in binding to low affinity FcγRs comparedto individual IgG molecules. For example, compared to IVIg, construct 5*displayed approximately 170 fold increased affinity FcγRIIa (H131variant), 55 fold increased affinity for FcγRIIb.

Example 10—Inhibition of Phagocytosis in THP-1 Monocytic Cells

Construct 5* and IVIg were tested in a model of phagocytosis.

Phagocytosis is the process by which cells (phagocytes) engulf solidparticles such as bacteria, to form an internal vesicle known as aphagosome. In the immune system, phagocytosis is a major mechanism usedto remove pathogens and cell debris. Monocytes and macrophages are amongthe cells specialized in clearing opsonized (antibody coated) particlesfrom the immune system through phagocytosis, a mechanism largelydependent on FcγR mediated engagement. However, in autoimmune diseases,phagocytes can become activated leading to the detrimental release ofpro-inflammatory cytokines or the phagocytosis of other critical cellsin the body. IVIg, containing pooled, polyvalent, IgG antibodiesextracted from the plasma of over one thousand blood donors, is used totreat autoimmune disease.

In this assay system, fluorescently labeled antibody-coated latex beads,a mimic of opsonized bacteria or viruses, were fed to THP-1 cells andallowed to be phagocytosed in the presence and absence of construct 5*and IVIg. At the end of the incubation period, any external fluorescencewas quenched with trypan blue, and the amount of intracellularfluorescence quantified by flow cytometry. All groups were normalized totheir non-treated control (THP-1 cells and latex beads only). Resultsare representative of two separate experiments.

As shown in FIG. 17, the phagocytosis of opsonized beads by THP-1monocytic cells is inhibited by treatment with both IVIg and construct5*, but the IC50 value for construct 5* is approximately 100-fold lowerthan for IVIg. This suggests that an Fc construct of the invention,e.g., construct 5*, can be used to treat autoimmune indications, as wellas other indications that are treatable using IVIg.

The invention claimed is:
 1. An Fc construct comprising: a). a firstpolypeptide having the formula A-L-B; wherein i). A comprises a first Fcdomain monomer; ii). L is a linker; and iii). B comprises a second Fcdomain monomer; b). a second polypeptide having the formula A′-L′-B′;wherein i). A′ comprises a third Fc domain monomer; ii). L′ is a linker;and iii). B′ comprises a fourth Fc domain monomer; c). a thirdpolypeptide that comprises a fifth Fc domain monomer; and d). a fourthpolypeptide that comprises a sixth Fc domain monomer; wherein A and A′combine to form a first Fc domain, B and the fifth Fc domain monomercombine to form a second Fc domain, and B′ and the sixth Fc domainmonomer combine to form a third Fc domain; the first polypeptidecomprises the same amino acid sequence as the second polypeptide, andthe third polypeptide comprises the same amino acid sequence as thefourth polypeptide; the second Fc domain monomer comprises a differentamino acid sequence than the fifth Fc domain monomer, and the fourth Fcdomain monomer comprises a different amino acid sequence than the sixthFc domain monomer, and each of the first, second, third, fourth, fifth,and sixth Fc domain monomers comprises a human IgG1 CH2 domain monomerand a human IgG1 CH3 domain monomer, wherein each CH3 domain monomer hasno more than 10 single amino acid substitutions to promote complementarydimerization of Fc domain monomers.
 2. The Fc construct of claim 1,wherein each of the CH3 domain monomers of the first and third Fc domainmonomers comprises a complementary dimerization selectivity module thatpromote dimerization between the first Fc domain monomer and the thirdFc domain monomer, each of the CH3 domain monomers of the second andfifth Fc domain monomers comprises a complementary dimerizationselectivity module that promote dimerization between the second Fcdomain monomer and the fifth Fc domain monomer, and/or each of the CH3domain monomers of the fourth and sixth Fc domain monomers comprises acomplementary dimerization selectivity module that promote dimerizationbetween the fourth Fc domain monomer and the sixth Fc domain monomer. 3.The Fc construct of claim 2, wherein the complementary dimerizationselectivity modules of the first and third Fc domain monomers eachcomprise reverse charge mutations in at least two positions within aring of charged residues at the interface between the CH3 domainmonomers of the first and third Fc domain monomers.
 4. The Fc constructof claim 3, wherein the reverse charge mutations in at least twopositions are K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D,K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E (all positions byEU Kabat numbering system).
 5. The Fc construct of claim 2, wherein thecomplementary dimerization selectivity modules of the first and third Fcdomain monomers comprise quadruple reverse charge mutations in fourpositions at the interface between the CH3 domain monomers of the firstand third Fc domain monomers.
 6. The Fc construct of claim 5, whereinthe quadruple reverse charge mutations combine any pair of doublemutations selected from K409D/D399K, K392D/D399K, E357K/K370E,D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, and D356K/K439E (allpositions by EU Kabat numbering system).
 7. The Fc construct of claim 2,wherein one of the complementary dimerization selectivity modules of thesecond and fifth Fc domain monomers comprises an engineered protuberanceand the other of the complementary dimerization selectivity modules ofthe second and fifth Fc domain monomers comprises an engineered cavity.8. The Fc construct of claim 2, wherein one of the complementarydimerization selectivity modules of the fourth and sixth Fc domainmonomers comprises an engineered protuberance and the other of thecomplementary dimerization selectivity modules of the fourth and sixthFc domain monomers comprises an engineered cavity.
 9. The Fc constructof claim 2, wherein the complementary dimerization selectivity modulesof the second and fourth Fc domain monomers each comprise an engineeredprotuberance and the complementary dimerization selectivity modules ofthe fifth and sixth Fc domain monomers each comprise an engineeredcavity.
 10. The Fc construct of claim 9, wherein the engineeredprotuberance comprises at least one mutation selected from the groupconsisting of S354C, T366W, T366Y, T394W, T394F, and F405W and theengineered cavity comprises at leastone mutation selected from the groupconsisting of Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394S(all positions by EU Kabat numbering system).
 11. The Fc construct ofclaim 2, wherein the complementary dimerization selectivity modules ofthe second and fourth Fc domain monomers each comprise an engineeredcavity and the complementary dimerization selectivity modules of thefifth and sixth Fc domain monomers each comprise an engineeredprotuberance.
 12. The Fc construct of claim 11, wherein the engineeredcavity comprises at least one mutation selected from the groupconsisting of Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394Sand the engineered protuberance comprises at least one mutation selectedfrom the group consisting of S354C, T366W, T366Y, T394W, T394F, andF405W (all positions by EU Kabat numbering system).
 13. The Fc constructof claim 1, wherein the Fc construct contains no more than three Fcdomains.
 14. The Fc construct of claim 1, wherein one or more linker insaid Fc construct is a spacer.
 15. A pharmaceutical compositioncomprising a substantially homogenous population of the Fc construct ofclaim 1 and one or more pharmaceutically acceptable carriers orexcipients.
 16. The Fc construct of claim 2, wherein the CH3 domainmonomers of the first and the third Fc domain monomers dimerize and eachof the complementary dimerization selectivity modules of the first andthird Fc domain monomers comprises reverse charge mutations in at leasttwo positions within a ring of charged residues at the interface betweenthe CH3 domain monomers of the first and the third Fc domain monomers;wherein the CH3 domain monomers of the second and the fifth Fc domainmonomers dimerize, the complementary dimerization selectivity module ofthe second Fc domain monomer comprises an engineered protuberance, andthe complementary dimerization selectivity module of the fifth Fc domainmonomer comprises an engineered cavity; and wherein the CH3 domainmonomers of the fourth and sixth Fc domain monomers dimerize, thecomplementary dimerization selectivity module of the fourth Fc domainmonomer comprises an engineered protuberance, and the complementarydimerization selectivity module of the sixth Fc domain monomer comprisesan engineered cavity.
 17. The Fc construct of claim 2, wherein the CH3domain monomers of the first and the third Fc domain monomers dimerize,the complementary dimerization selectivity module of the first Fc domainmonomer comprises an engineered protuberance, and the complementarydimerization selectivity module of the third Fc domain monomer comprisesan engineered cavity; wherein the CH3 domain monomers of the second andthe fifth Fc domain monomers dimerize and each of the complementarydimerization selectivity modules of the second and fifth Fc domainmonomers comprises reverse charge mutations in at least two positionswithin a ring of charged residues at the interface between the CH3domains of the second and the fifth Fc domain monomers; and wherein theCH3 domain monomers of the fourth and sixth Fc domain monomers dimerizeand each of the complementary dimerization selectivity modules of thefourth and sixth Fc domain monomers comprises reverse charge mutationsin at least two positions within a ring of charged residues at theinterface between the CH3 domains of the fourth and sixth Fc domainmonomers.
 18. The Fc construct of claim 2, wherein the CH3 domainmonomers of the first and third Fc domain monomers dimerize, thecomplementary dimerization selectivity module of the first Fc domainmonomer comprises an engineered cavity, and the complementarydimerization selectivity module of the third Fc domain monomer comprisesan engineered protuberance; wherein the CH3 domain monomers of thesecond and the fifth Fc domain monomers dimerize and each of thecomplementary dimerization selectivity modules of the second and fifthFc domain monomers comprises reverse charge mutations in at least twopositions within a ring of charged residues at the interface between theCH3 domains of the second and the fifth Fc domain monomers; and whereinthe CH3 domain monomers of the fourth and sixth Fc domain monomersdimerize and each of the complementary dimerization selectivity modulesof the fourth and sixth Fc domain monomers comprises reverse chargemutations in at least two positions within a ring of charged residues atthe interface between the CH3 domains of the fourth and the sixth Fcdomain monomers.
 19. The Fc construct of claim 1, wherein the amino acidsequences of the second and fifth Fc domain monomers differ by two aminoacids, and the amino acid sequences of the fourth and sixth Fc domainmonomers differ by two amino acids.
 20. The pharmaceutical compositionof claim 15, wherein the substantially homogenous population is 85%homogenous.
 21. The Fc construct of claim 1, wherein each of the first,second, third, fourth, fifth, and sixth Fc domain monomers furthercomprises a hinge domain or functional fragment thereof.
 22. The Fcconstruct of claim 1, wherein each of the first, second, and third Fcdomains are capable of binding to an Fc receptor.
 23. A pharmaceuticalcomposition comprising a substantially homogenous population of the Fcconstruct of any of claims 1-12, 13, 14, 16, 20, 21 and 22 and one ormore pharmaceutically acceptable carriers or excipients.
 24. A method ofreducing immune cell activation of the immune response in a subject,said method comprising administering to said subject the Fc construct ofany of claims 1-12, 13, 14, 16, 20, 21 and 22.